Induction of differential stress resistance and uses thereof

ABSTRACT

This invention relates to methods of inducing differential stress resistance in a subject with cancer by starving the subject for a short term, administering a cell growth inhibitor to the subject, or reducing the caloric or glucose intake by the subject. The induced differential stress resistance results in improved resistance to cytotoxicity in normal cells, which, in turn, reduces cytotoxic side-effects due to chemotherapy, as well as improved effectiveness of chemotherapeutic agents.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.12/058,600, filed Mar. 28, 2008, which issued on Jul. 3, 2012 as U.S.Pat. No. 8,211,700, which claims priority to U.S. ProvisionalApplication Ser. No. 60/908,636, filed Mar. 28, 2007, and U.S.Provisional Application Ser. No. 60/942,561, filed Jun. 7, 2007, thecontents of which are incorporated herein by reference in theirentirety.

FUNDING

The present invention was made, at least in part, with the financialsupport of NIH/NIA grants AG20642 and AG025135. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention, in general, relates to methods for treatingcancer. In particular, the present invention provides methods forenhancing the effectiveness of chemotherapy by inducing differentialstress resistance in normal cells and cancer cells via short-termstarvation, cell growth inhibitors, or reduced caloric or glucoseintake.

BACKGROUND OF THE INVENTION

Until recently, the treatment of cancer has been largely focused on thedevelopment of therapeutic agents or techniques that kill cancer cells.For example, most chemotherapeutic drugs work by impairing mitosis (celldivision), effectively targeting fast-dividing cells. As these drugscause damage to cells they are termed cytotoxic. Some drugs work bycausing cells to undergo apoptosis (so-called “cell suicide”).Unfortunately, scientists have yet to be able to locate specificfeatures of malignant and immune cells that would make them uniquelytargetable (barring some recent examples, such as the Philadelphiachromosome as targeted by imatinib). This means that other fast dividingcells such as those responsible for hair growth and for replacement ofthe intestinal epithelium (lining) are also affected.

Because chemotherapy affects cell division, both normal and cancerouscells are susceptible to the cytotoxic effects of chemotherapeuticagents. Success of conventional chemotherapeutic regiment is based onthe principle that tumors with high growth fractions (such as acutemyelogenous leukemia and the lymphomas, including Hodgkin's disease) aremore sensitive to chemotherapy because a larger proportion of thetargeted cells are undergoing cell division at any given time. Thisstrategy often results in undesirable side-effects such as hair loss andnormal tissue/organ damage. It also has severe limitations on the dosageof chemotherapeutic agents that can be administered to a patient, thus,limiting the effective range of chemotherapy.

SUMMARY OF THE INVENTION

The present invention provides a novel approach to cancer therapy byproviding a method to differentially enhance the resistance of normalcells to chemotherapeutic agents, thereby, improving the effectivenessof chemotherapeutic agents in killing cancerous cells. By making normalcells more resistant to chemotherapeutic agents, a patient's tolerancefor cytotoxicity is improved, which, in turn, also improves theeffectiveness of chemotherapy.

More specifically, in one aspect, the invention features methods ofinducing differential stress resistance in a subject with cancer. Onemethod comprises starving the subject for 24-60 (e.g., 48) hours andadministering to the subject a chemotherapy agent. The method mayfurther comprise administering to the subject a cell growth inhibitor.

Another method of the invention comprises administering a cell growthinhibitor to the subject and administering to the subject a chemotherapyagent. For example, by using a cell growth inhibitor, the serumconcentration of IGF-I in the subject may be reduced by 75-90%.

Another method of the invention comprises reducing the caloric intake orthe glucose intake by the subject and administering to the subject achemotherapy agent. For example, the caloric intake may be reduced by10-100%, and the blood glucose concentration in the subject may bereduced by 20-50%.

In another aspect, the invention features methods of contacting a cancercell with a chemotherapy agent and methods of increasing resistance of anon-cancer cell to a chemotherapy agent. One method comprises starvingthe cell for 24-60 (e.g., 48) hours and contacting the cell with achemotherapy agent. The method may further comprise contacting the cellwith a cell growth inhibitor.

Another method of the invention comprises contacting the cell with acell growth inhibitor and contacting the cell with a chemotherapy agent.

Another method of the invention comprises cultivating the cell in amedium with reduced serum, IGF-I, or glucose concentration andcontacting the cancer cell with a chemotherapy agent. For example, theserum concentration in the medium may be reduced by 10-90%, the IGF-Iconcentration in the medium may be reduced by 10-100%, and the glucoseconcentration in the medium may be reduced by 20-50%.

A chemotherapy agent may be a DNA alkylating agent, oxidant, ortopoisomerase inhibitor. Examples of chemotherapy agents include, butare not limited to, methyl methanesulfonate, cyclophosphamide,etoposide, doxorubicin, and menadione. Examples of cancer include, butare not limited to, glioma, neuroblastoma, pheochromocytoma, andprostate cancer.

A cell growth inhibitor inhibits, e.g., IGF-I, IGF-IR, GH, Akt, Ras,Tor, or Erk. Examples of cell growth inhibitors include, but are notlimited to, IGFBPs, IGF-R blocking antibodies, and small moleculeinhibitors such as octreotide.

The above-mentioned and other features of this invention and the mannerof obtaining and using them will become more apparent, and will be bestunderstood, by reference to the following description, taken inconjunction with the accompanying drawings. These drawings depict onlytypical embodiments of the invention and do not therefore limit itsscope.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. A) DSR of short-term starvation (STS) against acute H₂O₂ ormenadione treatments. Survival of wild type (DBY746), sch9/aktΔ,RAS2^(val19), sch9/aktΔras2Δ and sch9/aktΔRAS^(val19) after a 48-hourstarvation and exposure to H₂O₂ (400 mM, 60 min) and to menadione (1 mM,30 min). The cultures were diluted in K-phosphate buffer to an initialOD₆₀₀ 1 for both treatments. Serial dilutions were spotted onto YPDplates and incubated at 30° C. for 2-3 days. This experiment wasrepeated at least 3 times with similar results. A representativeexperiment is shown. B, C) DSR against chronic CP and MMS treatments inmixed cultures. sch9/aktΔ and sch9/aktΔRAS2^(val19) were mixed andincubated for 2 hours shaking at 30° C. The initial sch9/aktΔ:sch9/aktΔRAS2^(val19) ratio as measured by growth on selective media was 25:1.Mixed cultures were treated with either CP (0.1 M) or MMS (0.01%).Viability was measured by quantifying colony-forming units (CFUs) every24 hours by plating onto appropriate selective media. This experimentwas repeated at least 2 times with similar results. A representativeexperiment is shown. D) DSR against chronic CP treatment. Wild type(DBY746), sch9/aktA, RAS2^(val19), sch9/aktA and sch9/aktΔRAS2^(val19)stains were individually grown and treated with CP (0.1 M). Viabilitywas measured by the same method as in the mixed culture experiment.

FIG. 2. DSR against a chemotherapeutic drug (CP) in primary rat mixedglial cells and rat glioma cell lines. A) Glucose restriction inducedDSR. Cells were incubated in either low glucose (0.5 g/L) (STS) ornormal glucose (1.0 g/L), supplemented with 1% serum for 24 hours.Viability was determined by the ability to reduce MTT following CPtreatment (dose response range: 6-12 mg/ml) (n=9). B) Serum restrictioninduced DSR. Cells were incubated in medium with either 1% (STS) or 10%FBS for 24 hours. Cytotoxicity was determined by measuring the relativelevel of LDH released following CP treatment (15 mg/ml) (n=12). C) Lackof IGF-I induced DSR. Cells were incubated in medium with 1% serum andrhIGF-I (100 ng/ml) for 48 hours. Cytotoxicity was determined bymeasuring the relative level of LDH released following CP treatment (12mg/ml) (n=21). D) aIR3 induced DSR. Cells were incubated in DMEM/F12with 1% serum and neutralizing anti-IGF-IR monoclonal antibody aIR3 (1μg/ml) for 24 hours. Cytotoxicity was determined by measuring therelative level of LDH released following CP treatment (15 mg/ml) (n=12).All data presented as mean±SD. P-values were calculated by the Student'st-test (* p<0.05, ** p<0.01).

FIG. 3. A) Differential stress resistance in A/J mice after STS andoctreotide treatment. All mice received an i.v. injection of 80 mg/Kgetoposide (Eto) on day 7. The different groups were treated as follows:Gr. 1 (35 mice): pre-treatment with a 1 mg/Kg/day octreotide (OCT) for 4days+48-hour STS (day 4-6)+treatment with 80 mg/Kg Eto on day7+post-treatment with 1 mg/Kg/day octreotide (OCT) on days 8-11. Gr. 2(16 mice): 48-hour STS on days 4-6+treatment with 80 mg/Kg Eto on day 7.Gr. 3 (17 mice): pre-treatment with 1 mg/Kg/day octreotide (OCT) for 4days+treatment with 80 mg/Kg Eto on day 7+post-treatment with 1mg/Kg/day octreotide (OCT) on days 8-11. Gr. 4 (23 mice): treatment with80 mg/Kg Eto on day 7. B) Percentage weight loss (a measure of toxicity)after STS and etoposide treatment. C) Differential stress resistance inCD1 mice after STS. All mice (5 mice/group) received an i.v. injectionof 110 mg/Kg etoposide (Eto), after a 60-hour starvation. The toxicity,evaluated by percentage of survival, is shown. P values were calculatedby Peto's log rank test: (P<0.0001). D) Percentage weight loss (ameasure of toxicity) after STS and etoposide treatment. ** day at whichall mice died of toxicity.

FIG. 4. Survival of neuroblastoma (NXS2)-bearing mice after chemotherapytreatment. All mice were inoculated i.v. with 200,000 NXS2 cells/mouseon day 4. The different groups were treated as follows: A) Gr. 1:Control (8 mice)=i.v. inoculation with NSX2 tumor cells on day 4. Gr. 2:OCT (8 mice)=pre-treatment with 1 mg/Kg/day octreotide (OCT) for 4 daysbefore and after tumor inoculum. Gr. 3: OCT/STS/OCT (8mice)=pre-treatment with 1 mg/Kg/day OCT before tumor cellinoculum+48-hour STS on days 4-6+post-treatment with 1 mg/Kg/day OCT ondays 8-11. Gr. 4: OCT/STS/Eto/OCT (8 mice)=pre-treatment with 1mg/Kg/day OCT for 4 days before tumor cell inoculum+48-hour STS on day4-6+i.v. injection of 80 mg/Kg etoposide (Eto) on day 7+post-treatmentwith 1 mg/Kg/day OCT on days 8-11. Gr. 5: STS (8 mice)=48-hour STS ondays 4-6. Gr. 6: STS/Eto (7 mice)=48-hour STS on days 4-6+i.v. injectionof 80 mg/Kg etoposide (Eto) on day 7. B) Gr. 7: Eto (6 mice)=i.v.injection of 80 mg/Kg etoposide (Eto) on day 7. Gr. 8:OCT/Eto/OCT=pre-treatment with 1 mg/Kg/day OCT for 4 days before tumorcell inoculum+i.v. injection of 80 mg/Kg etoposide (Eta) on day7+post-treatment with 1 mg/Kg/day OCT on days 8-11. Statistics: P Gr. 4vs Gr. 1<0.0001, P Gr. 4 vs Gr. 3<0.0001, P Gr. 6 vs Gr. 1=0.14, P Gr. 8vs Gr. 1=0.38, P Gr. 7 vs Gr. 1=0.99, P Gr. 4 vs Gr. 6=0.01, P Gr. 8 vsGr. 5=0.66. Survival of mice was monitored daily. P values werecalculated by Peto's log rank test.

FIG. 5. A) OCT/STS/OCT treated mice and B) control mice shown afteretoposide treatment.

FIG. 6. A, B) Differential stress resistance (DSR) against chroniccyclophosphamide and methyl methanesulfonate treatments in mixed yeastcultures. sch9/aktΔ and sch9/aktΔ RAS2^(val19) cells were inoculated inSDC medium at OD=0.1 and incubated at 30° C. with shaking. 24 hourslater (OD˜10), sch9/aktΔ and sch9/aktΔ RAS2^(val19) were mixed andincubated for 2 hours at 30° C. with shaking. The initialsch9/akt:sch9/aktΔ RAS2^(val19) ratio, measured by growth on selectivemedia, was 25:1. Mixed cultures were then treated with either CP (0.1 M)or MMS (0.01%). Viability was measured every 24 hours by plating ontoappropriate selective media that allows the distinction of the 2strains. Data from 3 independent experiments are shown as mean±SD.

FIG. 7. A) Resistance to high-dose chemotherapy in A/J mice after STSand/or octreotide treatment. Mice were treated as follows: Gr. 1 (35mice): pre-treatment with 1 mg/kg/day octreotide for 4 days+48-hour STS(day 4-6)+treatment with 80 mg/kg Eto on day 7+post-treatment with 1mg/kg/day octreotide on days 8-11. Gr. 2 (16 mice): 48-hour STS on days4-6+treatment with 80 mg/kg Eto on day 7. Gr. 3 (17 mice): pre-treatmentwith 1 mg/kg/day octreotide for 4 days+treatment with 80 mg/kg Eto onday 7+post-treatment with 1 mg/kg/day octreotide on days 8-11. Gr. 4 (23mice): treatment with 80 mg/kg Eto on day 7. B) Percent weight loss ofA/J mice (a measure of toxicity) after STS and Eto treatment. C)Resistance to high-dose chemotherapy in CD1 mice after STS. All mice (5mice/group) received an i.v. injection of 110 mg/kg etoposide (Eto),after a 60-hour starvation. The toxicity, evaluated by percent survival,is shown. P values were calculated by Peto's log rank test: (P<0.0001).D) Percent weight loss of CD1 mice (a measure of toxicity) after STS andEto treatment. ** day at which all mice died of toxicity. E) Resistanceto high-dose chemotherapy in athymic (Nude-nu) mice after STS. All micereceived an i.v. injection of 100 mg/kg Eto, after a 48-hour starvation.The toxicity, evaluated by percent survival, is shown. F) Percent weightloss (a measure of toxicity) of athymic (Nude-nu) after STS andetoposide treatment. G) Resistance to high-dose chemotherapy in LIDmice. All mice received an i.p. injection of 500 mg/kg Cyclophosphomideand were single caged throughout the experiment. The toxicity, evaluatedby percent survival, is shown (p<0.002). H) Percent weight loss (ameasure of toxicity) of LID mice after Cyclophosphamide treatment. I)Survival fraction of STS treated and untreated mice after etoposideinjection. Mice from 3 different genetic backgrounds (A/J, CD1 and Nude)were injected with etoposide with or without STS pre-treatment. Alltreatments with STS have been combined and compared with all treatmentswithout STS and shown as percent survival after etoposide injection.Also, the percent survival of each treatment after etoposide injectionis compared and shown.

FIG. 8. Survival of neuroblastoma (NXS2)-bearing mice. All mice wereinoculated i.v. with 200,000 NXS2 cells/mouse on day 4. The differentgroups were treated as follows: A) Gr. 1: Control (16 mice)=i.v.inoculation with NSX2 tumor cells on day 4. Gr. 2: Oct (8mice)=pre-treatment with 1 mg/kg/day octreotide for 4 days before andafter tumor injection on day 4. Gr. 3: Oct/STS/Oct (8mice)=pre-treatment with 1 mg/kg/day Oct before tumor cell injection onday 4+48-hour STS on days 4-6+post-treatment with 1 mg/kg/day Oct ondays 8-11. Gr. 4: Oct/STS/Eto/Oct (8 mice)=pre-treatment with 1mg/kg/day Oct for 4 days before tumor cell injection+48-hour STS on day4-6+i.v. injection of 80 mg/kg etoposide (Eto) on day 7+post-treatmentwith 1 mg/kg/day Oct on days 8-11. Gr. 5: Oct/Eto/Oct (8mice)=pre-treatment with 1 mg/kg/day Oct for 4 days before tumor cellinjection+i.v. injection of 80 mg/Kg etoposide (Eto) on day7+post-treatment with 1 mg/kg/day Oct on days 8-11. Gr. 6: STS (8mice)=i.v. inoculation with NSX2 tumor cells on day 4+48-hour STS ondays 4-6. Gr. 7: STS/Eto (16 mice)=i.v. inoculation with NSX2 tumorcells on day 4+48-hour STS on clays 4-6+i.v. injection of 80 mg/kgetoposide (Eto) on day 7. Gr. 8: Eto (6 mice, *2 deaths caused by theinjection procedure)=i.v. inoculation with NSX2 tumor cells on day4+i.v. injection of 80 mg/kg etoposide (Eto) on day 7. B) Effect ofoctreotide on etoposide cytotoxicity in NXS2 neuroblastoma cells. NXS2cells treated with different concentrations of etoposide (1-3 microM) inthe presence or absence of octreotide (10 and 50 microM) for 72 hourswere harvested by scraping, washed with complete medium, and incubatedwith trypan blue for 1 minute at 37° C. Viability was determined bycounting the cells with a contrast phase microscope. The proportion ofdead (or living) cells was calculated by dividing the number of dead (orliving) cells by the total number of cells per field. C) The DSR model:oncogenes prevent cells from entering into a protective maintenance modein response to starvation and low IGF-1 signaling. One of the hallmarksof cancer cells is the ability to grow or remain in a growth moderegardless of external regulatory signals including IGF-1R, Ras, Akt andmTor.

FIG. 9. A) Oct/STS/Eto/Oct group and B) control group shown after Etotreatment (day 7).

FIG. 10. A, B) Survival of STS-treated yeast cells deficient in Sch9and/or Ras2 (sch9Δ, and sch9Δras2Δ), and cells overexpressing Sch9 orexpressing constitutively active RAS2^(val19) (SCH9, RAS2^(val19),sch9ΔRAS2^(val19), and tor1ΔRAS2^(val19)) after treatment with H₂O₂ ormenadione. 24 hours after the initial inoculation (OD=0.1) in SDCmedium, cultures were washed, resuspended and incubated in water for 48hours with shaking. At day 3, cells were treated with either H₂O₂ for 30min, or menadione for 60 min. Serial dilutions (up to 1,000-fold) of thetreated cultures were spotted onto YPD plates and incubated for 2-3 daysat 30° C. This experiment was repeated at least 3 times with similarresults. A representative experiment is shown. C) DSR against chronic CPtreatment. Wild type (DBY746), RAS2^(val19), sch9Δ and sch9ΔRAS2^(val19)strains were inoculated at OD=0.1, grown separately in glucose media,and treated with CP (0.1 M) 24 hours after initial inoculation.Viability was measured as colony forming units (CFU) at 24 and 48 hours.

FIG. 11. A) In vitro STS effect on differential stress response (DSR) tocyclophosphamide treatments. Primary rat glial cells and the C6 ratglioma cells were grown to 70% confluency and then incubated in eitherlow glucose (0.5 g/L) (STS) or normal glucose (1.0 g/L), supplementedwith 1% serum for 24 hours followed by cyclophosphamide (12 mg/ml)treatment. Cytotoxicity was measured by LDH release. Data represented asmean±SD. p-values were calculated using Student's t-test (** p<0.005).B) Phosphorylation of Erk1/2 in response to starvation conditions in 5cancer cell lines. Rat glioma and human neuroblastoma cell lines (FIG.11A) were starved by preincubation in glucose- and serum-free media for17 hours (STS), or were kept in glucose- and serum-free media for 16hours followed by a 1 hour treatment with serum (1% FBS). Western blotsshow Erk1/2 phosphorylation.

FIG. 12. 24 hr treatment with IGF-I sensitizes cortical neurons but notin PC12 cells to oxidative toxicity. Primary rat cortical neurons orPC12 cells incubated in 1% serum and 4 g/L glucose were treated for 24hr with vehicle, 100 μM of paraquat, 100 ng/ml of IGF-I followed by 100μM of paraquat, or 100 ng/ml of IGF-1 alone and were then subjected toMTT reduction activity assay to assess cell viability. Data from 4independent experiments are normalized to control and expressed as themean±S.E.M.

FIG. 13. Comparison of the long-term survival of mice from the Eto groupthat survived the initial toxicity and mice from the Oct/STS/Eto/Octgroup. Statistical evaluation was done using Kaplan-Meier curves andlog-rank test.

FIG. 14. A mouse from, the (A) control group and (B) liver-IGF-1 deleted(LID) group shown after cyclophosphamide treatment.

FIG. 15. Similar pathways regulate longevity and resistance to stress inyeast and mice.

FIG. 16. The IGF-I, Ras and Akt pathways, whose down-regulation regulateresistance to damage and aging in different model systems (FIG. 15),play central roles in mitosis and cancer.

FIG. 17. A, B) Survival of STS-treated yeast cells deficient in Sch9/Aktand/or Ras2 (sch9/aktΔ, and sch9/aktΔ ras2Δ), and cells overexpressingSch9/Akt or expressing constitutive active RAS2^(val19) (Sch9/Akt,RAS2^(val19), sch9/aktΔ RAS2^(val19), and tor1Δ RAS2^(val19)) aftertreatment with H₂O₂ or menadione. 24 hours after the initial inoculation(OD=0.1) in SDC medium, cultures were washed, resuspended and incubatedin water for 48 hours with shaking. At day 3, cells were treated witheither H₂O₂ for 30 min, or menadione for 60 min. Serial dilution (up to1,000-fold) of the treated cultures were spotted onto YPD plates andincubated for 2-3 days at 30° C. This experiment was repeated at least 3times with similar results. A representative experiment is shown. C)Differential stress resistance (DSR) against chronic cyclophosphamideand methyl methanesulfonate (MMS) treatments in mixed yeast cultures.sch9/aktΔ and sch9/aktΔ RAS2^(val19) cells were inoculated in SDC mediumat OD=0.1 and incubated at 30° C. with shaking. 24 hours later (OD˜10),sch9/aktΔ and sch9/aktΔ RAS2^(val19) were mixed and incubated for 2hours at 30° C. with shaking. The initial sch9/aktΔ:sch9/aktΔRAS2^(val19) ratio, measured by growth on selective media, was 25:1.Mixed cultures were then treated with either CP (0.1 M) or MMS (0.01%).Viability was measured every 24 hours by plating onto appropriateselective media that allows the distinction of the 2 strains. Data from3 independent experiments are shown as mean±SD. D) DSR against chronicCP treatment. Wild type (DBY746), RAS2^(val19), sch9/aktΔ andsch9/aktΔRAS2^(val19) strains were inoculated at OD=0.1, grownseparately in glucose media, and treated with CP (0.1 M) 24 hours afterinitial inoculation. Viability was measured as colony forming units(CFU) at 24 and 48 hours.

FIG. 18. Rates of survival and metastases in the LID-TRAMP model. Micewere analyzed at the time of death. Survival curves for the two strainsare shown. There was no significant difference in the survival ratebetween male LID-TRAMP and L/L-TRAMP mice. The hazard ratio of maleLID-TRAMP mice to male L/L-TRAMP mice was 0.736. There was no differencein rate of metastasis; however, IGF-I levels were 10% of TRAMP in theLID-TRAMP model.

FIG. 19. Levels of GH-IGF axis analytes in the LID TRAMP cohort. Shownare the endogenous elevations of IGF-I and IGFBP-3 in the TRAMP modeland the dramatic reductions in IGF-I in the LID model.

FIG. 20. A/J mice, weighing 18-20 g, were starved for 48 hours (STS),and followed by a single intravenous injection of high dose doxorubicin(16 mg/kg). The toxicity, evaluated by (A) percent survival and (B)daily weight measurements, are shown.

FIG. 21. Resistance to high-dose chemotherapy in LID mice. All micereceived an i.v. injection of 100 mg/kg etoposide and were single cagedthroughout the experiment. The toxicity, evaluated by (A) percentsurvival and (B) daily weight measurements, are shown. p-value wascalculated by log rank test (p=0.06, n=10).

FIG. 22. Resistance to multiple treatments with doxorubicin. Doxorubicinwas intravenously administered at 20 mg/kg on Day 0 and 28 mg/kg on Day22 (n=5). The toxicity, evaluated by (A) percent survival and (B) dailyweight measurements, are shown. p-value was calculated by log rank test(p<0.05).

FIG. 23. Inhibiting ERK1/2 protects neurons against oxidative stress.Cortical neurons from E18 rat embryos were cultured onto 96-well plates(A) or glass coverslips in 6-well plates (B). On 10-14 DIV, the neuronswere pre-treated with MEK1/ERK1/2 inhibitors U0126 or SL327. Oxidativestress was induced by hydrogen peroxide or menadione. Cell viability wasmeasured with MTT survival assay (A) or live/dead assay (B). C) % deathwas calculated from the number of dead (red) and live (green) cells.

DETAILED DESCRIPTION OF THE INVENTION

In the present invention, it has been unexpectedly discovered that thedeletion of SCH9/AKT genes from cells and/or short-term starvation (STS)treatment has a differential cyto-protective effect on normal cells ascompared with cancer or cancer-like cells expressing constitutivelyactively Ras2^(val19) (the cyto-protective effect may be up to 10,000times more effective). Further, reduction of IGF-I/IGF-IR signaling orSTS protected primary glial cells but not 6 different rat and humanglioma and neuroblastoma cancer cell lines against chemotherapy. LiverIGF-I deficient (LID) mice were also protected against high dosechemotherapy confirming the in vitro results and providing a candidatedrug target to replace starvation. STS in combination with thesomatostatin analogue octreotide provided complete protection to micebut did not protect injected cancer cells against high dosechemotherapy. The present invention thus provides evidence for theefficacy of short-term starvation and/or lowering IGF-IR signaling inthe protection of normal but not cancer cells against chemotherapy,radiotherapy or any other toxic treatment or environment.

One method of the invention involves starving a subject with cancer for24-60 (e.g., 30-55, 35-50, 40-45, and 48) hours and administering to thesubject a chemotherapy agent, thereby inducing differential stressresistance in the subject.

By “starving” is meant subjecting a cell or subject to reduced or nonutrients.

As used herein, a “subject” refers to a human or animal, including allmammals such as primates (particularly higher primates), sheep, dog,rodents (e.g., mouse or rat), guinea pig, goat, pig, cat, rabbit, andcow. In a preferred embodiment, the subject is a human. In anotherembodiment, the subject is an experimental animal or animal suitable asa disease model.

As used herein, “cancer” refers to a disease or disorder characterizedby uncontrolled division of cells and the ability of these cells tospread, either by direct growth into adjacent tissue through invasion,or by implantation into distant sites by metastasis. Exemplary cancersinclude, but are not limited to, primary cancer, metastatic cancer,carcinoma, lymphoma, leukemia, sarcoma, mesothelioma, glioma, germinoma,choriocarcinoma, prostate cancer, lung cancer, breast cancer, colorectalcancer, gastrointestinal cancer, bladder cancer, pancreatic cancer,endometrial cancer, ovarian cancer, melanoma, brain cancer, testicularcancer, kidney cancer, skin cancer, thyroid cancer, head and neckcancer, liver cancer, esophageal cancer, gastric cancer, intestinalcancer, colon cancer, rectal cancer, myeloma, neuroblastoma,pheochromocytoma, and retinoblastoma. Preferably, the cancer is glioma,neuroblastoma, pheochromocytoma, or prostate cancer.

Chemotherapy agents are antineoplastic drugs used to treat cancer,including alkylating agents, anti-metabolites, plant alkaloids andterpenoids, topoisomerase inhibitors, anti-tumour antibiotics,monoclonal antibodies, oxidants, hormones, and the like. Preferablechemotherapy agents are methyl methanesulfonate, cyclophosphamide,etoposide, doxorubicin, and menadione. Other chemotherapy agents areknown in the art, some of which are described below.

By short-term starvation, differential stress resistance (DSR) isinduced in a subject with cancer, i.e., while the resistance of cancercells to a chemotherapy agent is reduced or unchanged, the resistance ofnon-cancer cells (e.g., normal cells) to the chemotherapy agent isconcomitantly increased.

DSR may also be induced by administering a cell growth inhibitor to asubject with cancer. A “cell growth inhibitor” inhibits the growth of acell. For example, such inhibitors (e.g., IGFBPs, IGF-R blockingantibodies, and small molecule inhibitors such as octreotide) mayinhibit IGF-I, IGF-IR, GH, Akt, Ras, Tor, or Erk by inhibiting geneexpression or protein activity. In some embodiments, the serumconcentration of IGF-I in the subject may be reduced by 75-90% (e.g.,80-85%), as compared to normal conditions. Other cell growth inhibitorsare known in the art, some of which are described below.

Another method of inducing DSR in a subject with cancer is via reducingthe caloric or glucose intake by the subject. In some embodiments, DSRmay be achieved by reducing the caloric intake by 10-100% (e.g., 20-90%,30-80%, 40-70%, 50-60%) or by reducing the blood glucose concentrationin the subject by 20-50% (e.g., 25-45%, 30-40%, and 45%), as compared tonormal conditions.

The methods of the invention may be combined to maximize DSR. Forexample, DSR may be induced in a subject with cancer by a combination ofshort-term starvation and administration of a cell growth inhibitor.

The methods described above can be used to improve the effectiveness ofcancer treatment. A subject to be treated may be identified in thejudgment of the subject or a health care professional, and can besubjective (e.g., opinion) or objective (e.g., measurable by a test ordiagnostic method). To treat a subject with cancer, DSR is induced inthe subject prior to the administration of an effective amount of achemotherapy agent by short-term starvation, administration of aneffective amount of a cell growth inhibitor, glucose, etc.

The term “treatment” is defined as administration of a substance to asubject with the purpose to cure, alleviate, relieve, remedy, prevent,or ameliorate a disorder, symptoms of the disorder, a disease statesecondary to the disorder, or predisposition toward the disorder.

An “effective amount” is an amount of a compound that is capable ofproducing a medically desirable result in a treated subject. Themedically desirable result may be objective (i.e., measurable by sometest or marker) or subjective (i.e., subject gives an indication of orfeels an effect).

A compound to be administered can be incorporated into pharmaceuticalcompositions. Such compositions typically include the compounds andpharmaceutically acceptable carriers. “Pharmaceutically acceptablecarriers” include solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. See, e.g., U.S. Pat. No. 6,756,196.Examples of routes of administration include parenteral, e.g.,intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal, and rectal administration.Solutions or suspensions used for parenteral, intradermal, orsubcutaneous application can include the following components: a sterilediluent such as water for injection, saline solution, fixed oils,polyethylene glycols, glycerine, propylene glycol or other syntheticsolvents; antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite;chelating agents such as ethylene diaminetetraacetic acid; buffers suchas acetates, citrates or phosphates; and agents for the adjustment oftonicity such as sodium chloride or dextrose. pH can be adjusted withacids or bases, such as hydrochloric acid or sodium hydroxide. Theparenteral preparation can be enclosed in ampoules, disposable syringes,or multiple dose vials made of glass or plastic.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.“Dosage unit form,” as used herein, refers to physically discrete unitssuited as unitary dosages for the subject to be treated, each unitcontaining a predetermined quantity of an active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The dosage required for treating a subject depends on the choice of theroute of administration, the nature of the formulation, the nature ofthe subject's illness, the subject's size, weight, surface area, age,and sex, other drugs being administered, and the judgment of theattending physician. Suitable dosages are in the range of 0.01-100.0mg/kg. Wide variations in the needed dosage are to be expected in viewof the variety of compounds available and the different efficiencies ofvarious routes of administration. For example, oral administration wouldbe expected to require higher dosages than administration by intravenousinjection. Variations in these dosage levels can be adjusted usingstandard empirical routines for optimization as is well understood inthe art. Encapsulation of the compound in a suitable delivery vehicle(e.g., polymeric microparticles or implantable devices) may increase theefficiency of delivery, particularly for oral delivery.

Methods similar to those described above may be used to contact a cancercell with a chemotherapy agent (e.g., to reduce resistance of the cancercell to the chemotherapy agent) and to increase resistance of anon-cancer cell to the chemotherapy agent. Such method may involve, forexample, starving the cell for 24-60 (e.g., 30-55, 35-50, 40-45, and 48)hours, and optionally, contacting the cell with a cell growth inhibitor;contacting the cell with a cell growth inhibitor; or cultivating thecell in a medium with reduced concentration of serum (e.g., by 10-90%,20-80%, 30-70%, 40-60%, and 50%), IGF-I (e.g., by 10-100%, 20-90%,30-80%, 40-70%, and 50-60%), or glucose (e.g., by 20-50%, 25-45%,30-40%, and 45%), as compared to normal conditions.

These methods can be used to identify candidate protocols, cell growthinhibitors, and chemotherapy agents for in vivo applications. Forexample, a protocol, cell growth inhibitor, or chemotherapy agent may betested using the methods described above. If the protocol, cell growthinhibitor, or chemotherapy agent leads to reduced or unchangedresistance of a cancer cell to a chemotherapy agent and increasedresistance of a non-cancer cell to a chemotherapy agent under the sameconditions, the protocol, cell growth inhibitor, or chemotherapy agentis identified to be a candidate for in vivo application of DSRinduction.

The following examples are intended to illustrate, but not to limit, thescope of the invention. While such examples are typical of those thatmight be used, other procedures known to those skilled in the art mayalternatively be utilized. Indeed, those of ordinary skill in the artcan readily envision and produce further embodiments, based on theteachings herein, without undue experimentation.

Example I A Starvation Response-Based Differential Stress ResistanceMethod to Enhance Cancer Treatment Material and Methods

Cell Lines

Primary mixed glial cells were obtained from the cerebral cortex of 1 to3 day old Sprague Dawley rat pups (Charles River) as described before.Cells cultured for 10-14 days in DMEM/F12 medium with 10% fetal bovineserum (FBS) were used in assays. C6, A10-85, 9L and RG2 rat glioma celllines and LN229 human glioma cell line and SH-SY5Y human neuroblastomacell line were maintained in DMEM/F12 medium with 10% FBS at 37° C.under 5% CO₂.

STS Treatments

Cells were seeded into 96-well microliter plates at 20,000-30,000cells/well and incubated for 2 days. Cells were washed with phosphatebuffered saline (PBS) prior to treatments as indicated in the text. Alltreatments were performed at 37° C. under 5% CO₂.

Glucose restriction was done by incubating cells in glucose free DMEM(Invitrogen) supplemented with either low glucose (0.5 gL) or normalglucose (1.0 gL) for 24 hours. Serum restriction was done by incubatingcells in DMEMIF12 with either 10% or 1% FBS for 24 hours. IGF-Itreatment was carried out by incubating cells for 48 hours in DMEM/F12with 1% FBS and rhIGF-I (100 n g/m1, ProSpec-Tany TechnoGene, Rehovot,Israel), which is shown to be within the IGF-I level range for middleage humans. To antagonize IGF-I receptor activity, cells were incubatedwith neutralizing monoclonal anti-IGF-IR antibody (aIR3, 1 pg/ml;Calbiochem) in DMEM/F12 1% FBS for 24 hours.

In Vitro Chemotherapeutic Treatment

A widely used chemotherapeutic drug (cyclophosphamide, CP, Sigma) wasused for in vitro studies. CP was prepared in DMEM/F12 with 1% FBS at 40mg/ml and was filter sterilized. The stock solution was stored at 4° C.for no longer than 2 weeks.

Following STS treatments, cells were incubated with varyingconcentrations of cyclophosphamide (6-15 mg/ml) for 10 hours in DMEM/F12with 1% FBS. Cytotoxicity was measured by either LDH release using theCytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) or theability to reduce methylthiazolyldiphenyltetrazolium bromide (MTT). %LDH release was determined with reference to the maximum and backgroundLDH release of control cells. MTT assay results were presented aspercentage of MTT reduction level of control cells.

Yeast Strains and Growth Conditions

Experiments were carried out in wild type DBY746 MTa, leu2-3,112,his3D1, trp1-289, ura3-52, GAL+, and isogenic strains lacking eitherSch9 (Fabrizio et al., 2001) or both Sch9 and Ras2 (Fabrizio et al.,2001). Wild type (DBY746) and sch9A strains expressing hyperactiveRas2^(val19) constructed by transformation with a centromericplasmidcontaining Ras2^(val19) (pRS406-Ras2^(val19), CEN URA3). Yeast cultureswere grown in liquid synthetic dextrose complete medium (SDC) with 2%glucose, supplemented with amino acids, adenine, as well as a four-foldexcess of tryptophan, leucine, histidine, uracil. Strains harboring thecentromeric plasmid containing Ras2^(val19) were always grown in theabsence of uracil to maintain selection.

Yeast Viability

Overnight cultures were diluted to OD₆₀₀ 0.1 into SDC medium. After 24hours (day I), the appropriate strains were mixed 1:1 based on OD₆₀₀ andincubated for 2 hours. The mixed cultures were then treated with eithercyclophosphamide (CP, 0.1 M) or methylmethionine sulphonium chloride(MMS, 0.01%, Sigma) in medium or water (STS). For treatment SDC, MMS wasintroduced directly into the mixed culture to a final concentration of0.01%. However, due to the high concentration of CP (0.1 M) used, CP wasdissolved directly into the medium. To do so, mixed cultures werecentrifuged for 5 minutes at 2,500 rpm and the media was collected, inwhich CP was dissolved to a concentration of 0.1 M. The mixed culturewas then resuspended in the CP-containing medium. For treatments inwater (STS), mixed cultures were centrifuged for 5 minutes at 2,500 rpmand the media was replaced with either distilled/sterile water ordrug-dissolved water. Viability was measured by quantifyingcolony-forming units (CFUs) every 24 hours by plating onto appropriateselective media. Viability of individual strains was measured using thesame method as for the mixed cultures.

DSR Assays in Yeast

Stress resistance against MMS, menadione (Sigma), and H₂O₂ (Sigma) wasmeasured by spotting serial dilutions of control and treated cells.Briefly, overnight cultures were diluted in 1 ml of liquid SDC mediumwith 2% glucose to an initial OD₆₀₀ 0.1 and incubated overnight shakingat 30° C. 24 hours later, aliquots of cells were washed and resuspendedin water for 48 hours (STS). Following STS, cultures were diluted toOD₆₀₀ 1 in K-phosphate buffer and treated with menadione (1 mM; pH7.4)or H₂O₂ (400 mM, pH6.0) for 60 and 30 minutes, respectively. Then,serial dilutions were spotted onto YPD plates and incubated at 30° C.for 2-3 days.

In Vivo Therapeutic Studies in Mice

The murine NX3IT28 cell line was generated by hybridization of theGD2-negative C1300 murine neuroblastoma cell line (A/J background) withmurine dorsal root ganglional cells from C57BL16J mice, as previouslydescribed. The NXS2 sublime was then created by the selection of NX3IT28cells with high GD2 expression.

Six-to-seven-week-old female NJ mice, weighing 15-18 g were purchasedfrom Harlan Laboratories (Harlan Italy, S. Pietro al Natisone, Italy)and housed in sterile enclosures under specific virus and antigen-freeconditions. All procedures involving mice and their care were reviewedand approved by licensing and ethical committee of the National CancerResearch Institute, Genoa, Italy, and by the Italian Ministry of Health.

N J mice were pretreated with 1 mg/kg/day doses of human octreotide(OCT, ProSpec-Tany TechnoGene, Rehovot, Israel) for 4 days given slowlythrough the tail vein in a volume of 100 p1 and then injectedintravenously with murine neuroblastoma NXS2 cell line (200,000/mouse),as previously described. After tumor cell inoculum, some groups ofanimals were starved for 48 hours and then i.v. treated with 80 mg/kg ofEtoposide Teva (Teva Pharma B.V., Mijdrecht, Holland), administered as asingle dose. Additional daily doses of OCT were administered for 4 daysafter chemotherapy. Control groups of mice without diet starvation andOCT treatment were also investigated.

Octreotide pre-treatment: 4 days, 1 mg/kg/day

NXS2: 200,000/mouse on day 4

STS: from day 4 to day 6 (after tumor cell inoculum)

Etoposide: 80 mg/kg on day 7

Octreotide post-treatment: days 8-11

To determine toxicity and efficacy, mice were monitored routinely forweight loss and general behavior. The animals were killed by cervicaldislocation after being anesthetized with xilezine (Xilor 2%, Bio98 Srl,Milan, Italy) when they showed signs of poor health, such as abdominaldilatation, dehydration, or paraplegia. Survival time was used as themain criterion for determining the efficacy of each treatment.

The statistical significance of differential survival betweenexperimental groups of animals was determined by Kaplan-Meier curves andlog-rank eeto test by the use of StatDirect statistical software(Camcode, Ashwell, UK).

In some experiments, four-week-old female CD1 mice (Harlan), weighing18-20 g were used to evaluate stress resistance after 60 hours ofstarvation. These animals, i.v. injected with 110 mg/kg etoposide, weremonitored routinely for weight loss and general behavior. Survival timewas used as the main criterion for determining the differential stressresistance after short-term starvation.

Experimental

The inventor's diligent studies in S. cerevisiae and those of others inworms, flies, and mice have uncovered a strong association between lifespan extension and resistance to multiple stresses. This resistance isobserved in long-lived yeast cells lacking the orthologs of the humanRas (RAS2) and Akt (SCH9/AKT) proto-oncogenes and in long-lived wormsand mice with reduced activity of homologs of the IGF-I receptor(IGF-IR), which functions upstream of Ras and Akt in mammalian cells,also implicated in many human cancers. This resistance is also observedin model systems in which the calorie intake is reduced by 30 to 100%.Based on the unexpected discovery of the role of Ras2 and Sch9/Akt inthe negative regulation of stress resistance together with theassociation between mutations that activate IGF-IR, Ras or Akt and manyhuman cancers, it was believed that normal but not cancer cells wouldrespond to starvation or down-regulation of growth hormone GH/IGF-Isignaling by entering a chemotherapy resistance mode. It was also notedthat one of the major phenotypic characteristics of malignant cells isthe hyperactivation of pathways including the IGF-IR, Ras and Aktpathways and the ability to grow or remain in a growth mode even in theabsence of growth factors.

To test whether constitutively active oncogenes or oncogene homologswould prevent the switch to a protective maintenance mode in response tostarvation, it was first determined whether acute starvation would be aseffective in increasing stress resistance as long-term calorierestriction (CR).

DSR studies in S. cerevisiae were performed. A short-term starvationparadigm was selected as well as the deletion of the SCH9/AKT and/orRAS2 genes, each of which mimics in part calorie restriction. It wasbelieved that the combination of these genetic manipulations withstarvation would maximize DSR. The combination of STS (switch fromglucose medium to water for 24-48 hours) with the deletion of SCH9/AKTor both SCH9/AKT and RAS2 homolog causes a 1,000- to 10,000-fold DSR inresponse to a 30- to 60-minute treatment with hydrogen peroxide ormenadione compared to cells expressing the constitutively activeRAS2^(val19) or cells lacking SCH9/AKT (sch9/akfA) but expressingRAS2^(val19) (sch9/aktΔ RAS2^(val19)) (FIG. 1A). STS of wild type cellsalso caused DSR relative to RAS2^(val19) expressing cells but thedifferential effect for wild type cells was lower in response to H₂O₂treatment and was not observed after treatment with the hydrogenperoxide and superoxide generating agent menadione (FIG. 1A). Thetypical increase of resistance to hydrogen peroxide after STS comparedto incubation in medium was between 10- and 100-fold for wild typecells. The objective for this experiment was to model in a simple systemthe effect of the combination of short-term starvation and a geneticapproach on differential stress resistance between normal and cancercells. The results showed that the expression of the oncogene-likeRAS2^(val19) prevented the 1,000- to 10,000-fold protection caused bythe combination of STS and inhibition of Sch9/Akt activity.

To test whether DSR would also occur after treatment of yeast withchemotherapy drugs, the effect of STS and SCH9/AKT mutations on thetoxicity caused by alkylating agents methyl methanesulfonate (MMS) andcyclophosphamide (CP, a widely used chemotherapy drug) was studied. As amodel for the effect of STS and/or IGF-I inhibition on advanced stagecancer in which cancer cells were mixed with normal cells, mutantslacking SCH9/AKT with mutants lacking SCH9/AKT but also expressingRAS2^(val19) were mixed in the same flask at a 25:1 ratio. They werethen exposed to chronic treatment with CP or MMS. The viability of thetwo populations could be assessed by the ability of each population togrow on plates containing different selective media. Of theapproximately 10 million sch9/aktΔRAS2^(val19) cells mixed with 250million sch9/aktΔ, none of the sch9/aktΔRAS2^(val19) cells survived a48-hour treatment with 0.01% MMS (FIG. 1B). By contrast, the greatmajority of sch9/aktΔ survived this treatment (FIG. 1B). Similar resultswere obtained with mixed sch9/aktΔRAS2^(val19)/sch9/aktΔ culturestreated with cyclophosphamide (CP) (FIG. 10). A similar experiment inwhich each cell type was treated with CP separately was also performed,and a similar differential stress resistance between cells expressingRAS2^(val19) and cells lacking SCH9/AKT was observed (FIG. 1D). Notably,a 48-hour treatment with CP was toxic to both RAS2^(val19) and wild typecells (FIG. 1D) suggesting that the lack of SCH9/AKT causes a majorincrease in protection relative to RAS2^(val19) expressing cells butalso wild type cells. These results confirm that DSR obtained byaltering gene expression is effective in protecting normal cells but notcancer-like cells against chemotherapy.

To test the efficacy of the starvation-based DSR method on mammaliancells, primary rat mixed glial cells (astrocytes+5-10% microglia) orthree different rat glioma tumor cell lines in medium containing eithernormal (1 g/L) or low (0.5 g/L) glucose (1 g/L. glucose is within thenormal human blood glucose range. A 0.5 g/L glucose concentration can bereached during starvation) were incubated and then treated with thechemotherapy drug CP. Whereas 80% of glial cells were resistant to 12mg/ml CP in the presence of 0.5 g/L glucose, only 20% of the cellssurvived this treatment in 1 g/L glucose (FIG. 2A). The differentialstress resistance between the two concentrations of glucose (0.5 and 1g/L) was observed starting at 6 mg/ml CP but became much more pronouncedat 12 mg/ml CP (FIG. 2A). By contrast, the lower glucose concentrationdid not affect the resistance of either C6, A10-85, or RG2 glioma cellsto the same doses of CP (FIG. 2A). The lower glucose concentrationactually increased the toxicity of CP to RG2 glioma cells at 6 and 8mg/ml doses (FIG. 2A).

A similar DSR effect was obtained with a different form of starvationachieved by reducing the serum concentration. Treatment with 15 mg/ml CPwas toxic to primary glial cells in 10% serum but the switch to 1% serumcaused a major reduction in toxicity (FIG. 2B). Instead, the sameconcentration of CP was as toxic to C6 glioma cells in 10% serum as itwas in 1% serum (FIG. 213).

In S. cerevisiae experiments it was shown that the deletion of SCH9/AKTprotects whereas the constitutive activation of Ras2 (RAS2^(val19))sensitizes the yeast cells to chemotherapy drugs. Since mammalian Rasand Akt function downstream of the IGF-I receptor, and considering therole of the IGF-I pathway in regulating stress resistance, the effect ofIGF-I and of an antibody against IGF-IR on DSR was also tested.Treatment with 100 ng/ml IGF-I (in the low IGF-I level range for normalhuman adults) caused a 4-fold increase in the toxicity ofcyclophosphamide to primary mixed glia but only caused a minor increasein the toxicity of CP to C6 glioma cells (FIG. 2C). Furthermore,pre-incubation with an IGF-IR antagonist antibody protected primary gliabut only provided a small protection of two of the three glioma linestested against CP toxicity (FIG. 2D). These results in normal glia andin glioma cell lines are consistent with those in yeast cells and showedthat short-term starvation and/or drugs that down-regulateGH/IGF-I/Ras/Akt signaling can protect normal cells much moreeffectively than cancer cells against chemotherapy.

In an in vivo test, mice were treated with high doses of chemotherapy incombination with STS and/or a GH/IGF-I lowering therapy. For thispurpose etoposide, a widely used chemotherapy drug which damages DNA bymultiple mechanisms and displays a generalized toxicity profile rangingfrom myelosuppression to liver and neurologic damage, was selected.Unusually high doses of etoposide (80-110 mg/Kg) were administered aftera GH/IGF-I lowering treatment, period of starvation or both. In humans,a third of this concentration of etoposide (30-45 mg/Kg) is consideredto be a high dose and therefore in the upper allowable range.

To reduce GH/IGF-I A/J mice were pre-treated for four days with thegrowth hormone release inhibitor somatostatin analogue octreotide. Thispre-treatment was followed by etoposide administration. A sub-group ofmice were also starved for 48 hours before treatment with etoposide. Themice pre-treated with octreotide received this treatment for 4additional days after chemotherapy. Whereas 80 mg/Kg etoposide killed43% of control (Eto, n=23, 2 experiments) and 29% of octreotidepre-treated mice (OCT/Eto, n=17), in two separate experiments none ofthe mice treated with octreotide and also pre-starved for 48 hours diedafter 80 mg/kg etoposide treatment (OCT/STS/Eto/OCT, n=35) and only oneof the mice that were only starved (STS/Eto, n=16) died after etoposidetreatment (FIG. 3A). Remarkably, STS/octreotide pre-treated mice, whichlost 25% of the weight during the 48 hours of starvation, regained allthe weight in the four days after etoposide treatment (FIG. 3B) whereasin the same period the control mice lost approximately 20% of the weight(FIG. 3B). Non-STS pre-treated mice also showed reduced mobility andruffled hair.

The effect of STS alone on the protection of mice of another geneticbackground (CD1) was also tested. To determine whether an extended STSstrategy can be effective against a higher concentration of chemotherapydrugs 110 mg/Kg etoposide was administered and also the starvationperiod was increased to 60 hours. Based on the experiments withoxidative stress, it was determined that this period is the maximum STSthat provides protection. Longer starvation periods can weaken theanimals and have the opposite effect. This concentration of etoposidekilled all the control mice (Eto 110) but none of the STS pre-treatedmice (STS/Eto 110, n=5) (FIG. 3C). As for the A/J mice, CD1 STSpre-treated mice, which lost 40% of the weight during the 60 hours ofstarvation, regained all the weight in the week after the etoposidetreatment and showed no sign of toxicity (FIG. 3D). In summary, out of56 mice from two genetic backgrounds that were starved before etoposidetreatment, only one mouse from the STS only group and none of the micefrom the STS/octreotide pre-treated group died. By contrast, out of the45 mice treated with etoposide alone or etoposide and octreotide, 20died of toxicity.

These results are consistent with the yeast and glia/glioma data showingincreased resistance to chemotherapy toxicity in response to starvation.In mice, octreotide alone was not sufficient to protect againstetoposide toxicity and virtually all the protection was due instead toSTS. Notably, octreotide treatment was sufficient to protect miceagainst the superoxide generator paraquat.

To determine whether the differential stress resistance observed inyeast and mammalian cells would also occur in mice, the survival of miceinjected with cancer cells was followed. A particularly aggressive tumorline (NXS2) was selected, which models a common childhood cancer:neuroblastoma. The NXS2 neuroblastoma line induces consistent andreproducible metastases in a pattern which resembles the situationobserved in neuroblastoma patients at advanced stages of disease.Experimental metastases in the liver, kidneys, adrenal gland, andovaries were observed after 25-30 days of the inoculation with 200,000NXS2 cells (Table 1) as previously described. Although the tumordevelopment and survival of STS/etoposide treated mice was notsignificantly different from that of controls (Gr. 6 vs Gr: 1 p=0.14),half of the pre-starved mice were alive at a point when thenon-etoposide treated mice were all dead (FIG. 4A, Table 1) suggestingthat STS only partially protects cancer cells against etoposide. Thus,several or many chemotherapy cycles in combination with STS may berequired for the effective killing of all the metastasized cancer cells.

TABLE 1 Median Survival Adrenal Hemorrhagic survival Range Groups LiverKidneys Ovaries gland ascites (days) (days) Gr. 1 Control 16/16 14/1613/16  3/16 16/16 32 32-38 Gr. 2 OCT 8/8 8/8 8/8 2/8 8/8 33 31-37 Gr. 3OCT/STS/OCT 8/8 5/8 6/8 1/8 8/8 31 27-33 Gr. 4 OCT/STS/Eto/OCT  5/8* 5/8*  5/8*  0/8*  6/8*  56* 46-90 Gr. 5 STS 8/8 5/8 7/8 1/8 8/8 3026-35 Gr. 6 STS/Eto 14/14  7/14  5/14  0/14 14/14 44 35-60 Gr. 7 Eto 1/3*  1/3*  1/3*  0/3*  1/3*  90* 87-90 Gr. 8 OCT/Eto/OCT  0/3**  0/3** 0/3**  0/3**  0/3**  90** 90-90

Remarkably, none of the octreotide/STS/etoposide injected with NXS2cells died until day 46, and at day 90, 25% of these mice were stillalive (FIG. 4, Table 1) (Gr. 4 vs Gr. 1 p=0.0001). Mice in theNXS2/etoposide and octreotide/NXS2/etoposide groups also did not developtumors until day 80 (Table 1) but, as described above, 50-65% of themwere killed by the initial dose of etoposide (FIGS. 3A and 413). The 25%cancer-free rate at 90 days of the octreotide/STS/NXS2/etoposide-treatedmice (FIG. 4A) together with the major postponement of metastasis anddeath in the remaining 75% of these mice (Table 1) suggest that thistreatment is a highly effective strategy to kill cancer cells whileprotecting the organism. Considering that the STS/etoposide/NXS2-treatedgroup did not survive as long as the group that also included octreotide(survival of Gr. 6 vs. Gr. 4 p=0.01), these results indicate thatoctreotide contributes to killing NXS2 cells, possibly by inducingapoptosis as shown by others. In fact, somatostatin receptors areexpressed in neuroblastoma cells but also in breast and colon cancercells. Thus, octreotide may play a dual role in enhancing protection ofnormal cells while enhancing the death of cancer cells. However, in theabsence of short-term starvation, octreotide alone did not protect miceagainst NXS2-dependent death (FIG. 4), suggesting that it is thesynergism between etoposide and octreotide that is effective in killingcancer cells. The 90-day cancer free survival of the 3octreotide/etoposide-treated mice (Table 1) that were not killed by theinitial dose of etoposide (FIG. 4B) is consistent with such an effectiverole of the combination of high doses of chemotherapy and octreotide incuring metastatic cancer.

The conserved DSR response between normal cells (yeast and mammaliancells) and cancer-like or cancer cells demonstrates that it is possibleto protect the organism much more effectively than under normal feedingconditions while maintaining the toxicity of chemotherapy to tumorcells. The basis for this is the existence of a “maintenance mode”entered in response to starvation for the purpose of investing theremaining energy resources in protecting every cell and tissue. Inyeast, worms and mice starvation or the genetic manipulation ofstarvation response pathways causes a major increase in protectionagainst multiple stresses including heat shock and oxidative damage. Inmammals, starvation causes a reduction in IGF-I levels which isassociated with increased stress resistance. IGF-I is only one of thefactors involved in the starvation response, but are among the majornegative regulators of the entry into a stress resistant mode. Althoughthe role of calorie restriction or reduced activity of theGH/IGF-I/RAS/AKT axis in the stress resistance of primates is unknown,studies in monkeys and humans suggest that calorie restriction can haveeffects similar to those observed in simple eukaryotes and mice.

One of the most surprising discovery of this invention is the ability ofmice that have been starved for 48 hours to gain back the 25-40% weightlost during starvation even in the presence of doses of etoposide thatcause a 20-30% weight loss and kill over 40% of the control mice. Thishigh resistance to a drug that damages the DNA of dividing cells and isparticularly toxic to blood cells would be consistent with the entry ofmost or all of the normally dividing cells into a highly protective cellcycle arrested mode in response to the 48 hour starvation. Sinceetoposide is known to be rapidly excreted (up to 90% within 48 hours inhumans), such “protective mode” may only need to last for a few days.

Chemotherapy treatment has relied mainly on one or a combination ofseveral DNA damaging agents such as etoposide, cyclophosphamide, anddoxorubicin. Although these agents are supposedly much more toxic tocancer cells than to normal cells, the in vitro studies show thatcyclophosphamide, for example, can be as or more toxic to primary glialcells as it is to glioma cells. This implies that the combination ofmultiple chemotherapy drugs causes massive damage to normal cells,especially at high doses. Thus, it is particularly important to applystrategies such as the one presented here and aimed at reducing oreliminating the side effects of normal doses of chemotherapy or limitingthe toxicity of very high doses of chemotherapy that are effectiveagainst metastatic cancers.

Notably, the differential stress resistance of mammalian cells to thealkylating agents cyclophosphamide by the starvation-response methodswas less than 10-fold whereas starved yeast lacking SCH9/AKT reached a1,000-10,000-fold higher resistance to menadione, hydrogen peroxide,MMS, and cyclophosphamide compared to cancer-like RAS2^(val19)expressing yeast cells (FIGS. 1 and 2). Furthermore, the 10,000-folddifferential toxicity in yeast was obtained after only 30 minutes withhydrogen peroxide compared to the several days required for thedifferential toxicity of MMS or cyclophosphamide. Although toxicmolecules such as hydrogen peroxide or menadione are probably notsuitable for human treatments, these results suggest that the selectionof novel chemotherapy drugs in combination with DSR can result in agreatly more effective cancer treatment and may lead to treatments thatcan be carried out in minutes to hours. The ability to reach a10,000-fold differential toxicity between cancer cells and normal humancells would most likely lead to the cure of many cancers including thoseat advanced metastatic stages. Considering the remarkable resultsobtained with the aggressive NXS2 neuroblastoma line that was injectedin mice it is reasonable to expect novel drugs and conditions that canyield results close to the 10,000-fold DSR.

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Example II Starvation And IGF-I Reduction Protect Normal but not CancerCells Against High Dose Chemotherapy

The inventor's studies in S. cerevisiae and those of others in worms,flies, and mice have uncovered a strong association between life spanextension and resistance to multiple stresses. This resistance isobserved in long-lived yeast cells lacking the orthologs of the humanRas (RAS2) and Akt (SCH9/AKT) proto-oncogenes and in long-lived wormsand mice with reduced activity of homologs of the IGF-I receptor(IGFIR), which functions upstream of Ras and Akt in mammalian cells, andis also implicated in many human cancers. This resistance is alsoobserved in model systems in which the calorie intake is reduced by atleast 30%.

To test whether constitutively active oncogenes or oncogene homologs canprevent the switch to a protective maintenance mode in response tostarvation, it was first determined whether acute starvation would be aseffective in increasing stress resistance as it has been shown forlong-term calorie restriction (CR). Such long-term CR strategy would notbe appropriate for human chemotherapy treatments since it requiresseveral months to be effective.

DSR studies were first performed in S. cerevisiae. A short-termstarvation paradigm as well as the deletion of the SCH9/AKT and/or RAS2genes were selected, each of which mimics in part calorie restrictionand was shown in previous studies to cause high resistance to oxidativestress. It was believed that the combination of these geneticmanipulations with starvation would maximize DSR. The combination of STS(switch from glucose medium to water at day 1 [OD=9-10] and incubationin water for 24-48 hours) with the deletion of SCH9/AKT or both SCH9/AKTand RAS2 increased resistance to a 30- to 60-minute treatment withhydrogen peroxide or menadione a 1,000- to 10,000-fold compared to cellsexpressing the constitutively active oncogene homolog RAS2^(val19) orcells lacking SCH9/AKT (sch9/aktΔ) but expressing RAS2^(val19)(sch9/aktΔRAS2^(val19)) (FIG. 10A). The rationale for this experimentwas to model in a simple system the effect of the combination ofshort-term starvation and a genetic approach on the differentialprotection of normal and cancer cells. The results show that theexpression of the oncogene-like RAS2^(val19) prevents the1,000-10,000-fold protection caused by the combination of STS andinhibition of Sch9/Akt activity.

The effect of another oncogene homolog (SCH9/AKT) on resistance tooxidants was tested. As with RAS2^(val19), overexpression of SCH9/AKTsensitized yeast cells to both H₂O₂ and menadione was also tested (FIG.10B). Similarly to the effect of the deletion of RAS2 and SCH9/AKT, thedeletion of the homolog of TOR, another gene implicated in cancer,slightly increased the resistance to oxidants. Whereas the expression ofRAS2^(val1) completely reversed the protective effect of the deletion ofsch9/akt, it only had a minor effect on the reversal of the protectiveeffect of the tor1Δ (FIG. 10B). This is an important difference becauseit suggests that it may be risky to achieve DSR by inhibitingintracellular targets since inhibition of certain targets may alsoprotect cancer cells.

To test whether DSR would also occur after treatment of yeast withchemotherapy drugs, the effect of SCH9/AKT mutations on the toxicitycaused by alkylating agents methyl methanesulfonate and cyclophosphamide(CP, a widely used chemotherapy drug) was studied. As a model for theeffect of STS and/or IGF-I inhibition on metastatic cancer, mutantslacking SCH9/AKT were mixed in the same flask with mutants lackingSCH9/AKT but also expressing RAS2^(val19) at a 25:1 ratio and themixture was exposed to chronic treatment with CP or MMS. The monitoringof the viability of the two mixed populations was possible since eachpopulation can be distinguished by growth on plates containing differentselective media. Of the approximately 10 million sch9/aktΔRAS2^(val19)cells mixed with 250 million sch9/aktΔ, less than 5% of thesch9/aktΔRAS2^(val19) cells survived a 48-hour treatment with 0.01% MMS(FIG. 6A). By contrast, the great majority of sch9/aktΔ survived thistreatment (FIG. 6A). The combination of these mutations with STS wasalso effective against a yeast model for cancer cells and killed all theRAS2^(val19)-expressing cells by 24 hours. Similar results were obtainedwith mixed sch9/aktΔRAS2^(val19)/sch9/aktΔ cultures and cyclophosphamidetreatment (FIG. 6B). An experiment in which each cell type was treatedwith CP separately was also performed, and a similar differential stressresistance between cells expressing RAS2^(val19) and cells lackingSCH9/AKT was observed (FIG. 10C). These results confirm that mutationsin the Ras and Sch9/Akt and starvation are effective in protectingnormal cells but not yeast cells that model cancer cells (RAS2^(val19))against chemotherapy.

To test the efficacy of the starvation-based DSR method on mammaliancells, primary rat mixed glial cells (astrocytes+5-10% microglia) orthree different rat and one human glioma and one human neuroblastomacell lines were incubated in medium containing 1% serum and eithernormal (1 g/L) or low (0.5 g/L) glucose and then treated with thechemotherapy drug CP (1 g/L glucose is within the normal human bloodglucose range whereas a 0.5 g/L glucose concentration can be reached inhumans during starvation). The 1% serum concentration minimizes thecontribution of glucose from serum, which is approximately 1 g/L toavoid major differences in proliferation. Glia and glioma cells wereallowed to reach a 100% confluency. Whereas 80% of glial cells wereresistant to 12 mg/ml CP in the presence of 0.5 g/L glucose, only 20% ofthe cells survived this treatment in 1 g/L glucose (FIG. 2A). Thedifferential stress resistance between the two concentrations of glucose(0.5 and 1 g/L) was observed starting at 6 mg/ml CP but became much morepronounced at 12 mg/ml CP (FIG. 2A). By contrast, the lower glucoseconcentration did not affect the resistance of cancer cell linesincluding C6, A10-85, RG2 rat glioma, LN229 human glioma or humanSH-SY5Y neuroblastoma cells to 12-14 mg/ml CP (FIG. 2A). The lowerglucose concentration actually increased the toxicity of CP to RG2glioma cells at 6 and 8 mg/ml doses (FIG. 2A). To determine whether theDSR is affected by the high cell density, this experiment was repeatedwith cells that were only 70% confluent and similar results wereobtained (FIG. 11).

To determine whether the Ras/Erk pathway may be implicated in theunresponsiveness of the glioma and neuroblastoma cells above tostarvation the phosphorylation of Erk, which functions downstream ofRas, was measured in 10% serum or serum starved cells. Thephosphorylation data indicate that 2 of the 5 lines maintain a highlevel of Erk activity even after a 16-hour starvation (FIG. 11B), inagreement with the 30% frequency of Ras mutations in human cancers. Thereduction of Erk phosphorylation but inability to increase protection tocyclophosphamide in the other 3 lines (FIG. 2A) is consistent with ananti-resistance role of other common mutations in pro-growth pathwayssuch as the PTEN/PI3K/AKT pathway. Thus, it is believed that theconstitutive activation of any pro-growth pathway by a mutation wouldmake cancer cells unresponsive or much less responsive to thestarvation- or IGF-I reduction-dependent protection.

The experiments discussed above were performed in medium containing 1%glucose and different concentrations of glucose. The effect of reducingthe level of serum from the standard 10% to 1% on the toxicity ofhigh-dose cyclophosphamide was also tested. Treatment with 15 mg/ml CPwas toxic to primary glial cells in 10% serum but the switch to 1% serumcaused a reduction in toxicity (FIG. 2B). By contrast, the sameconcentration of CP was as toxic to C6 glioma cells in 10% serum as itwas in 1% serum (FIG. 2B).

In the S. cerevisiae experiments, it was showed that the deletion ofSCH9/AKT protects whereas the constitutive activation of Ras2(RAS2^(val19)) sensitizes the yeast cells to chemotherapy drugs. Sincemammalian Ras and Akt are major signal transduction proteins downstreamof the IGF-I receptor, and considering the role of the IGF-I pathway inregulating stress resistance, the effect of IGF-I and of an antibodyagainst IGF-IR on DSR was also tested. It was reasoned that primarycells would respond to the IGF-I-inhibiting treatment by increasingstress resistance whereas cancer cells, which often express oncogenesthat cause constitutive Ras and Akt activation, would not. Treatmentwith 100 ng/ml IGF-I (in the low IGF-I range for human adults) caused a3-fold increase in the toxicity of cyclophosphamide to primary mixedglia but did not increase the toxicity of CP to C6 glioma cells (FIG.2C). Furthermore, pre-incubation with an anti-IGF-IR antibody (aIR3)protected primary glia but not three glioma cell lines tested against CPtoxicity (FIG. 2D). A similar sensitizing role of IGF-I was observedwith primary rat cortical neurons but not the rat pheochromocytoma tumorPC12 cell line treated with oxidants (FIG. 12). These results in normalglia and in rat and human glioma and neuroblastoma cell lines areconsistent with those in yeast cells and support the belief thatshort-term starvation and/or drugs that down-regulate IGF-IR/Ras/Aktsignaling can protect normal cells much more effectively than cancercells against chemotherapy. Notably, this differential stress resistanceshould apply to the great majority of pro-growth oncogenic mutations andnot only to cancer cells with mutations in the Ras, Akt or Tor pathways.In fact, it worked with all the six cancer cell lines tested (FIG. 2),independently of the type of oncogenic mutations.

In an in vivo test, mice were treated with high dose chemotherapy incombination with STS and/or GH/IGF-I lowering strategies. For thispurpose, etoposide, a widely used chemotherapy drug which damages DNA bymultiple mechanisms and displays a generalized toxicity profile rangingfrom myelosuppression to liver and neurologic damage, was selected.Unusually high doses of etoposide (80-110 mg/kg) after a period ofstarvation, GH/IGF-I lowering treatment or both were administered. Inhumans, a third of this concentration of etoposide (30-45 mg/kg) isconsidered to be a high dose and therefore in the maximum allowablerange.

To reduce GH/IGF-I mice were pre-treated for four days with thesomatostatin analogue octreotide. This pre-treatment was followed byetoposide administration. A sub-group of mice were also starved for 48hours (STS) before treatment with etoposide. The mice pre-treated withoctreotide received this treatment for 4 additional days afterchemotherapy. Whereas 80 mg/kg etoposide killed 43% of control (Eto,n=23, 2 experiments) and 29% of octreotide pre-treated mice (OCT/Eto,n=17), none of the mice treated with octreotide and also pre-starved for48 hours died after 80 mg/kg etoposide treatment (Oct/STS/Eto/Oct, n=35)and only one of the mice that were only starved (STS/Eto, n=16) diedafter etoposide treatment (FIG. 7A). Remarkably, STS-octreotidepre-treated mice, which lost 20% of the weight during the 48 hours ofstarvation, regained all the weight in the four days after chemotherapy(FIG. 7B) whereas in the same period the control mice lost approximately20% of the weight (FIG. 7B). Control mice treated with etoposide showedsigns of toxicity including reduced mobility, ruffled hair and hunchedback posture (FIG. 9B) whereas Oct/STS/Oct pre-treated mice showed novisible signs of stress or pain following etoposide treatment (FIG. 9A).

The effect of STS alone on the protection of mice of another geneticbackground (CD1) was also tested. To determine whether an extended STSstrategy can be effective against a higher concentration of chemotherapydrugs 110 mg/kg etoposide was administered and the starvation period wasalso increased to 60 hours. Based on the experiments with oxidativestress, it was determined that this period is the maximum STS thatprovides protection. Longer starvation periods can weaken the animalsand have the opposite effect. This concentration of etoposide killed allthe control mice (Eto 110) but none of the STS pre-treated mice (STS/Eto110, n=5) (FIG. 7C). As with the A/J mice, pre-starved CD1 mice lost 40%of the weight during the 60 hours of starvation but regained all theweight in the week after the etoposide treatment and showed no visiblesign of toxicity (FIG. 7D).

The effect of the STS-based method was similar in athymic (Nude-nu)mice, which are widely used in cancer research because they allow thestudy of human tumors in the mouse model. Whereas 100 mg/kg etoposidekilled 56% of the nude mice and all the mice co-treated with octreotide,none of the STS/Eto/Oct or STS/Eto treated mice (48-hour starvation)died (FIG. 7E). As observed with the other two genetic backgrounds, thepre-starved mice gained weight during the period in which theEto-treated mice lost weight (FIG. 7F).

In summary, out of 70 mice from three genetic backgrounds that werestarved before etoposide treatment, only one mouse in the STS only groupand none of the mice in the STS/Oct group died (FIG. 7I). By contrast,out of the 63 mice treated with etoposide alone or etoposide andoctreotide, 34 died of toxicity. These results are consistent with theyeast and glia/glioma data showing increased resistance to chemotherapytoxicity in response to starvation or low IGF-I. In mice, octreotidealone was not sufficient to protect against etoposide toxicity andvirtually all the protection was due instead to STS. The discrepancybetween the effect of IGF-I in vitro and octreotide in vivo may be dueto the relatively modest effect of octreotide on the reduction ofcirculating IGF-I level.

To determine whether a much more severe reduction in IGF-I level thanthat achieved with octreotide can increase resistance to chemotherapy invivo as was shown in vitro, the resistance against a high dose ofcyclophosphamide of male and female LID mice, in which the liver IGF-Igene was conditionally deleted, resulting in a 75-90% reduced serumIGF-I concentration was studied. The LID mice treated with 500 mg/kgcyclophosphamide showed a remarkable improvement in resistance, with 30%mortality vs the 70% mortality for control mice (FIG. 7G, P<0.002).Furthermore, the LID mice lost an average of 10% of weight vs the 20%weight loss in control mice (FIG. 7H). The 70% surviving LID mice alsodid not show any signs of toxicity (FIG. 13B). Together with the invitro results with IGF-I and anti-IGF-IR antibodies and the establishedrole of starvation on the reduction of IGF-I levels, these data suggestthat the inhibition of IGF-IR signaling mediates part of the effects ofSTS on resistance to chemotherapy in vivo. Notably, IGF-IR antibodieshave been used successfully in a number of studies to reduce the growthor increase the death of cancer cells and are currently being evaluatedin clinical trials.

To determine whether the differential stress resistance observed inyeast and mammalian cells would also occur in mice, the survival of miceinjected with cancer cells was followed. A particularly aggressive tumorline (NXS2) that models neuroblastoma (NB), the most common extracranial solid tumor, and the first cause of lethality in pre-school agechildren, was selected. Advanced NB patients, who representapproximately 50% of the cases, show metastatic dissemination atdiagnosis, and have a long-term survival rate of only 20% in spite ofaggressive chemotherapy with autologous hematopoietic stem cell support.

The NXS2 neuroblastoma line in mice induces consistent and reproduciblemetastases in a pattern which resembles the clinical scenario observedin neuroblastoma patients at advanced stages of disease. Experimentalmetastases in the liver, kidneys, adrenal gland, and ovaries wereobserved after 25-30 days of the inoculation with 200,000 NXS2 cells(Table 2) as previously described.

TABLE 2 Frequency of metastases and survival in NXS2 injected mice (STS,Oct, Etoposide treatments) (Groups 1-8 represent the mice shown in FIG.8A). N^(o) Median Survival N^(o) toxicity Adrenal Hemorrhagic survivalRange Groups mice deaths Liver Kidneys Ovaries gland ascites (days)(days) Gr. 1 16  0/16 16/16 14/16 13/8   3/16 16/16 32 32-38 Control Gr.2 OCT 8 0/8 8/8 8/8 8/8 2/8 8/8 33 31-37 Gr. 3 8 0/8 8/8 5/8 6/8 1/8 8/831 27-33 OCT/STS/ OCT Gr. 4 8 0/8 8/8 8/8 6/8 0/8 6/8 56  46-130OCT/STS/ Eto/OCT Gr. 5 8 5/8  2/3*  2/3*  0/3*  0/3*  2/3* 130* 120-180OCT/Eto/ OCT Gr. 6 STS 8 0/8 8/8 5/8 7/8 1/8 8/8 30 26-35 Gr. 7 16  1/1615/15  9/15  9/15  0/15 14/15 44 35-60 STS/Eto Gr. 8 Eto 6 3/6 3/3 3/30/3 0/3 3/3 130   87-140A/J mice were i.v. inoculated with 200,000 NXS2 cells/mouse and treatedas described in Experimental Procedures. All mice were followed untildeath and necropsies were performed. Only 3 mice from group 7 and 8survived the initial etoposide treatment. The deaths caused by etoposidetoxicity in the early days that occurred in groups 6, 7, 8 were notconsidered in the calculation of median survival. *1 mouse alive at 180days.

The tumor development and survival of STS/Eto treated mice wassignificantly different from that of controls (Gr. 7 vs. Gr. 1 p<0.0001)(FIG. 8, Table 2) suggesting that STS alone provides strong protectionto the mouse but only partially protection of cancer cells againstetoposide. Based on these results, the use of STS alone would requireseveral or many chemotherapy cycles to obtain toxicity to cancer cellscomparable to that caused by high dose chemotherapy alone.

By contrast, none of the Oct/STS/Eto/Oct injected with NXS2 cells dieduntil day 46, at a point when all the controls had died of cancer (FIG.8, Table 2) (Gr. 4 vs. Gr. 1 p<0.0001). One mouse from this groupsurvived until day 130. The survival of Oct/NXS2/STS/Eto/Oct mice wasnot statistically significantly different from that of NXS2/Eto andOct/NXS2/Eto/Oct groups (Table 2) but, as described above, 50% of micenot protected with STS died of etoposide toxicity (FIGS. 7A and 8). Evenif the initial etoposide-dependent death is not considered, thelong-term survival of the octreotide/STS/NXS2/etoposide group was notsignificantly different from that of the NXS2/etoposide andsignificantly different but close to that of the Oct/NXS2/Eto/Oct group(FIG. 13). These results suggest that in combination with octreotide,STS protects the animal but not the cancer cells against chemotherapy.

Somatostatin analogues have been reported to promote anti-tumor activitythrough two distinct effects: direct actions, mediated by somatostatinreceptors, and indirect actions, independent of the receptors. Thesomatostatin/octreotide receptor-mediated effect includes inhibition ofcell cycle and growth factors effects, and induction of apoptosis. Incontrast, the indirect effects comprise inhibition of the release ofgrowth factors such as growth hormone and IGF-I.

To determine whether octreotide was acting directly on cancer cells, thetoxicity of etoposide to NXS2 cells cultured in vitro in the presence orabsence of octreotide was tested. Either 10 or 50 micromolar octreotidedid not sensitize NXS2 cells to etoposide treatment suggesting that itis not increasing the survival of the NXS2 injected mice by directlysensitizing the cells to etoposide (FIG. 8B). Because of the manystudies showing an anti-tumor growth and survival effect of lower GH andIGF-I or inhibition of the IGF-I and GET receptors, these resultssuggest that octreotide is improving long-term survival of STS treatedmice by its well established role in decreasing GH and IGF-I levels,although other effects cannot be ruled out. Notably octreotide and othersomatostatin analogues have been shown to have therapeutic effects in anumber of cancer. In the absence of short-term starvation, octreotidealone did not protect mice against NXS2-dependent death (FIG. 8),suggesting that it is the synergism between etoposide and octreotidethat is effective in killing tumor cells. These results also suggestthat octreotide pre-treatment before the injection of NXS2 cells doesnot affect the tumor growth.

The data above indicate that short-term starvation and/or reduction ofIGF-I/IGF-IR signalling protects normal cells and mice but not a varietyof cancer cells treated with chemotherapy drugs. While not wanting to bebound by the theory, it is believed that the basis for this is theexistence of a “maintenance mode” which cells enter in response tostarvation for the purpose of investing the remaining energy resourcesin protection of cells and tissues. In yeast, worms, and mice starvationor the genetic manipulation of starvation response pathways causes amajor increase in protection against multiple stresses including heatshock and oxidative damage. In mammals, starvation causes a reduction inIGF-I levels which is associated with increased stress resistance. IGF-Iis only one of the factors involved in the starvation response, but isamong the major negative regulators of the entry into a stress resistantmode. In this study, it was showed that LID mice which have a dramaticreduction in circulating IGF-I are protected against chemotherapytoxicity, although the protection is not as effective as that providedby short-term starvation. Notably, in preclinical studies IGF-I loweringand IGF-IR targeting strategies have been shown to be effective in thetreatment of multiple myelomas, prostate, breast and colon cancer inaddition to other cancers. Thus, the combination of IGF-IR targeting andhigh dose chemotherapy should be an effective strategy to kill cancercells, while preserving the health of the host.

Perhaps one of the most surprising findings of the present invention isthe ability of mice of 3 different genetic backgrounds that have beenstarved for 48 hours to gain back the 20-40% weight that were lostduring starvation even in the presence of doses of etoposide that causea 20-30% weight loss and kill over 40% of the control mice. These micealso showed no visible sign of toxicity in response to doses ofchemotherapy highly toxic to control animals. This high resistance to adrug that damages the DNA of dividing cells, particularly blood cells,would be consistent with the entry of most or all of the normallydividing cells into a high protection/cell cycle arrested mode inresponse to the 48-hour starvation. Since etoposide is rapidly excreted(up to 90% within 48 hours in humans), such “protective mode” may onlyneed to last for a few days.

Chemotherapy treatment often relies mainly on one or a combination ofseveral DNA damaging agents such as etoposide, cyclophosphamide, anddoxorubicin. Although these agents are supposedly much more toxic tocancer cells than to normal cells, the in vitro studies show thatcyclophosphamide, for example, can be as or more toxic to primary glialcells as it is to glioma cancer cells. This implies that the combinationof multiple chemotherapy drugs causes massive damage to normal cells,especially at high doses. Notably, the differential stress resistance ofmammalian cells to the alkylating agent cyclophosphamide by thestarvation-response methods was less than 10-fold whereas starved yeastlacking SCH9/AKT reached a 1,000-10,000-fold higher resistance tomenadione, hydrogen peroxide, MMS, and cyclophosphamide compared toRAS2^(val19) expressing yeast cells (FIGS. 6 and 2). Furthermore, the10,000-fold differential toxicity in yeast was obtained after only 30minutes with hydrogen peroxide compared to the several days required forthe differential toxicity of MMS or cyclophosphamide. Although toxicmolecules such as hydrogen peroxide or menadione are not suitable forhuman cancer treatments, these results suggest that the selection ofnovel chemotherapy drugs in combination with DSR can result in a morerapid and effective toxicity to cancer cells. The ability to reach a10,000-fold differential toxicity between cancer cells and normal humancells should lead to improved therapies for many cancers. Based on theresults obtained with the aggressive NXS2 neuroblastoma line that wasinjected in mice it is reasonable to attempt to identify novel drugs andconditions that can yield a much greater differential toxicity to normaland cancer cells.

The method discussed above covers the use of any drug, protein, peptide,or molecule that inhibits any component of the growth hormone releasinghormone, growth hormone, growth hormone receptor, IGF-I, IGF-I receptor,Ras, Akt axis or inhibits other growth factors or growth factorreceptors for the purpose of protecting normal human cells and humansubjects but not (or less) cancer and other cells against chemotherapy,radiotherapy, cancer treatment, or any other toxic treatment orenvironment.

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Timms, A. R., Muriel, W. & Bridges, B. A. A UmuD,C-dependent    pathway for spontaneous G:C to C:G transversions in stationary phase    Escherichia coli mut Y. Mutat Res 435, 77-80 (1999).-   15. Poole, C. J. et al. Epirubicin and cyclophosphamide,    methotrexate, and fluorouracil as adjuvant therapy for early breast    cancer. N Engl J Med 355, 1851-62 (2006).-   16. Brown-Borg, H. M., Rakoczy, S. G., Romanick, M. A. &    Kennedy, M. A. Effects of growth hormone and insulin-like growth    factor-I on hepatocyte antioxidative enzymes. Exp Biol Med 227,    94-104. (2002).-   17. Manetta, J. et al. Effect of training on the GH/IGF-I axis    during exercise in middle-aged men: relationship to glucose    homeostasis. Am J Physiol Endocrinol Metab 283, E929-36 (2002).-   18. Grunberg, S. M. Cyclophosphamide and etoposide for non-small    cell and small cell lung cancer. Drugs 58 Suppl 3, 11-5 (1999).-   19. Mistry, A. R. et al. DNA topoisomerase II in therapy-related    acute promyelocytic leukemia. N Engl J Med 352, 1529-38 (2005).-   20. Vinolas, N., Graus, F., Mellado, B., Caralt, L. & Estape, J.    Phase II trial of cisplatinum and etoposide in brain metastases of    solid tumors. J Neurooncol 35, 145-8 (1997).-   21. Kroger, N. et al. Busulfan, cyclophosphamide and etoposide as    high-dose conditioning therapy in patients with malignant lymphoma    and prior dose-limiting radiation therapy. Bone Marrow Transplant    21, 1171-5 (1998).-   22. Gronbaek, H. et al. Inhibitory effects of octreotide on renal    and glomerular growth in early experimental diabetes in mice. J    Endocrinol 172, 637-43 (2002).-   23. Hunter, S. J. et al. Comparison of monthly intramuscular    injections of Sandostatin LAR with multiple subcutaneous injections    of octreotide in the treatment of acromegaly; effects on growth    hormone and other markers of growth hormone secretion. Clin    Endocrinol (Oxf) 50, 245-51 (1999).-   24. Yakar, S. et al. Normal growth and development in the absence of    hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96,    7324-9 (1999).-   25. Ngo, T. H., Barnard, R. J., Tymchuk, C. N., Cohen, P. &    Aronson, W. J. Effect of diet and exercise on serum insulin, IGF-I,    and IGFBP-1 levels and growth of LNCaP cells in vitro (United    States). Cancer Causes Control 13, 929-35 (2002).-   26. Maccario, M. et al. Effects of 36-hour fasting on GHIIGF-I axis    and metabolic parameters in patients with simple obesity. Comparison    with normal subjects and hypopituitary patients with severe GH    deficiency. Int J Obes Relat Metab Disord 25, 1233-9 (2001).-   27. De Bernardi, B. et al. Disseminated neuroblastoma in children    older than one year at diagnosis: comparable results with three    consecutive high-dose protocols adopted by the Italian Co-Operative    Group for Neuroblastoma. J Clin Oncol 21, 1592-601 (2003).-   28. Matthay, K. K. et al. Treatment of high-risk neuroblastoma with    intensive chemotherapy, radiotherapy, autologous bone marrow    transplantation, and 13-cis-retinoic acid. Children's Cancer Group.    N Engl J Med 341, 1165-73 (1999).-   29. Lode, H. N. et al. Targeted interleukin-2 therapy for    spontaneous neuroblastoma metastases to bone marrow. J Natl Cancer    Inst 89, 1586-94 (1997).-   30. Zalatnai, A. & Schally, A. V. Treatment of    N-nitrosobis(2-oxopropyl)amine-induced pancreatic cancer in Syrian    golden hamsters with D-Trp-6-LH-RH and somatostatin analogue RC-160    microcapsules. Cancer Res 49, 1810-5 (1989).-   31. Susini, C. & Buscail, L. Rationale for the use of somatostatin    analogs as antitumor agents. Ann Oncol 17, 1733-42 (2006).-   32. Hejna, M., Schmidinger, M. & Raderer, M. The clinical role of    somatostatin analogues as antineoplastic agents: much ado about    nothing? Ann Oncol 13, 653-68 (2002).-   33. McPherson, A., Jr. The growth and preliminary investigation of    protein nucleic acid crystals for x-ray diffraction analysis. Met.    Biochem. Anal. 23, 249-345 (1976).

Example III Materials and Methods

Yeast Strains and Growth Conditions.

All the experiments were performed using yeast of DBY746 background.Knockout strains were prepared with standard PCR-mediated genedisruption protocol. Strains used in this study:

Strain Genotype DBY746 MATa, leu2-3, 112, his3Δ1, trp1-289, ura3-52,GAL⁺ sch9/aktΔ DBY746 sch9::URA3 ras2Δ DBY746 ras2::LEU2 tor1Δ DBY746tor1::HIS3 RAS2^(val19) DBY746 RAS2^(val19) (CEN, URA3)*tor1ΔRAS2^(val19) DBY746 ras2::LEU2 RAS2^(val19) (CEN, URA3)*sch9/aktΔRAS2^(val19) DBY746 sch9::URA3 RAS2^(val19) (CEN, URA3)*SCH9/AKT DBY746 SCH9 (CEN, URA3)* *pRS416-RAS2^(val19) was constructedby inserting 1.9 kb ClaI-HindIII fragment from pMF100 into pRS416.

Yeast cells were grown in minimal medium (SC) containing 2% glucose andsupplemented with amino acids as described previously. For short termstarvation (STS) treatment, day 1 cultures (24 hours after initialOD=0.1 inoculation) were washed 3 times with sterile distilled water,resuspended in water, and incubated at 30° C. with shaking for 48 hours(day 3).

Yeast Oxidative Stress Assay.

For oxidative stress resistance assays, day 3 cells were diluted to anOD₆₀₀ of 1 in K-phosphate buffer, pH 7.4, and treated with 0.2-1 mMmenadione for 1 hour. Alternatively, cells were diluted to an OD₆₀₀ of 1in K-phosphate buffer, pH 6 and treated with 200-400 mM of hydrogenperoxide for 30 minutes. Serial dilutions of untreated and treated cellswere spotted onto YPD plates and incubated at 30° C. for 2-3 days.

Yeast Viability Assay.

Overnight SDC cultures were diluted to OD₆₀₀ 0.1 into fresh SDC medium.After 24 hours (day 1), the appropriate strains were mixed 1:1 based onOD₆₀₀ (OD₆₀₀ of 10 each) and incubated for 2 hours at 30° C. withshaking. The mixed cultures were then treated with eithercyclophosphamide (CP, 0.1 M) or methyl methanesulfonate (MMS, 0.01%,Sigma). MMS was prepared in ddH₂O from stock solution and was diluteddirectly into the mixed culture to a final concentration of (v/v) 0.01%.However, due to the high concentration of CP (0.1 M) required, CPcrystals were dissolved directly into the medium. To do so, mixedcultures were centrifuged for 5 minutes at 2,500 rpm and the media wascollected, in which CP crystals were dissolved to a concentration of 0.1M. The mixed culture was then resuspended in the CP-containing medium.Viability was measured as colony-forming units (CFUs) every 24 hours byplating onto appropriate selective media. Viability of individualstrains was measured using the same method as for the mixed cultures.Relative survival shown was determined by the percentage of the ratiobetween the treated and untreated (control) cells.

Cell Culture Experiments.

Primary mixed glial cells were obtained from the cerebral cortices of 1to 3 day old Sprague Dawley rat pups (Charles River) as describedbefore. Cells cultured for 10-14 days in DMEM/F12 medium (Invitrogen)with 10% fetal bovine serum (FBS) were used in assays. Primary neuronsfrom embryonic day 18 Sprague-Dawley rat cerebral cortices weredissociated in neurobasal medium (Invitrogen) supplemented with 0.5 mML-glutamine, 25 μM L-glutamic acid and 2% B-27 and plated at 640cells/mm² in 96-well plates which were pre-coated with 10 microg/mlpoly-D-lysine dissolved in Borax buffer (0.15 M, pH 8.4). Neurons weremaintained at 37° C. in 5% CO₂ in neurobasal medium supplemented with13-27 and 0.5 mM L-glutamine for 4 days.

C6, A10-85, 9L and RG2 rat glioma cell lines and LN229 human glioma cellline, and SH-SY5Y human neuroblastoma cell line were maintained inDMEM/F12 medium with 10% FBS and the PC12 rat pheochromocytoma cell line(ATCC) was maintained in F12K medium supplemented with 15% horse serumand 2.5% fetal bovine serum at 37° C. under 5% CO₂.

STS Treatments of Mammalian Cells.

Primary glia, glioma or neuroblastoma cells were seeded into 96-wellmicrotiter plates at 20,000-30,000 cells/well and incubated for 2 days.Cells were washed with phosphate buffered saline (PBS) prior totreatments as indicated in the text. All treatments were performed at37° C. under 5% CO₂.

Glucose restriction was done by incubating cells in glucose free DMEM(Invitrogen) supplemented with either low glucose (0.5 g/L) or normalglucose (1.0 g/L) for 24 hours. Serum restriction was done by incubatingcells in DMEM/F12 with either 10% or 1% FBS for 24 hours. IGF-Itreatment was carried out by incubating cells for 48 hours in DMEM/F12with 1% FBS and rhIGF-I (100 ng/ml, ProSpec-Tany TechnoGene, Rehovot,Israel), which is shown to be within the IGF-I level range for middleage humans. To antagonize IGF-I receptor activity, cells were incubatedwith neutralizing monoclonal anti-IGF-1R antibody (aIR3, 1 microg/ml;Calbiochem) in DMEM/F12 1% FBS for 24 hours.

In Vitro Drug Treatments.

Cyclophosphamide (CP, Sigma) was used for in vitro chemotherapy studies.CP was prepared in DMEM/F12 with 1% FBS at 40 mg/ml and was filtersterilized. The stock solution was stored at 4° C. for no longer than 2weeks. Following STS treatments, cells were incubated with varyingconcentrations of cyclophosphamide (6-15 mg/ml) for 10 hours in DMEM/F12with 1% FBS. Glial cells have been reported to express cytochrome P450and thus capable of metabolizing the prodrug cyclophosphamide.Cytotoxicity was measured by either lactate dehydrogenase released usingthe CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega) or theability to reduce methylthiazolyldiphenyl-tetrazolium bromide (MTT). LDHreleased into the medium by lysed cells were measured with a 10-minuteenzymatic assay that converts a tetrazolium salt (INT) into a redformazan product. A 96-well based colorimetric assay measured the amountof the red formazan formed, which is proportional to the number of deadcells. % LDH release was determined with reference to the maximum andbackground LDH release of control cells.

MTT is reduced in the mitochondria (metabolically active cells) bymitochondrial reductase enzymes to form insoluble purple formazancrystals, which are solubilized by the addition of a detergent. Briefly,MTT was prepared at 5 mg/ml in PBS and was diluted in DMEM/F12 1% FBSmedia to a final concentration of 0.5 mg/ml for assays. Followingexperimental treatments, media was replaced with 100 μl of MTT and wasincubated for 3-4 hours at 37° C. Formazan crystals were dissolvedovernight (16 hours) at 37° C. with 100 μl lysis buffer ((w/v) 15% SDS,(v/v) 50% dimethylformamide). MTT assay results were presented aspercentage of MTT reduction level of treated cells to control cells.Absorbance was read at 490 nm and 570 nm for LDH and MTT assaysrespectively using a microplate reader SpectraMax 250 (MolecularDevices) and SoftMax Pro 3.0 software (Molecular Devices).

NXS2 neuroblastoma cells treated with different concentrations ofetoposide (1-3 μM) in presence or absence of octreotide (10 and 50 μM)for 72 hours were harvested by scraping, washed with complete medium,and incubated with trypan blue (0.04%; Sigma; St. Louis, Mo.) for 1minute at 37° C. The cells were then placed in a Burker chamber(Tecnovetro, Monza Milan, Italy) and counted by viewing with a contrastphase microscope (Olympus Optical Co LTD, Tokyo, Japan). Trypanblue-positive cells (i.e., dead cells), trypan blue-negative cells(i.e., living cells), and total cells were counted per microscope fieldat ×100 magnification (four fields were counted for each treatment). Theproportion of dead (or living) cells was calculated by dividing thenumber of dead (or living) cells by the total number of cells per field.

Primary rat neurons and PC12 cells were treated with IGF-1 and paraquatto determine the effect of IGF-1 on oxidative stress. Cortical neuronswere treated for 24 hours in Eagle's minimal essential medium(Invitrogen) supplemented with 21 mM glucose and 1% horse serum. PC12cells were plated at 5×10⁴ cells/well onto poly-D-lysine coated 96-wellplates and were grown for 24 hours in F12K 1% HS. Both types of cellswere then treated with either 100 μM of paraquat, IGF-1 (100 ng/ml)followed 30 minutes later by paraquat (100 μM) or IGF-1 (100 ng/ml)alone in appropriate media. Survival was determined by the MTT assay andpresented as percent ratio of treated to control.

Statistical analyses were performed using GraphPad Prism 4 software(GraphPad Software).

Western Blots.

Cells were seeded at 2×10⁶ cells/well in a 6-well culture plate andcultured in DMEM/F12 10% FBS for 1 day. Cells were washed with PBS twiceand glucose and serum starved by incubating in glucose-free DMEM for 16hours, followed by 1 hour of 10% FBS treatment or 1 additional hour ofstarvation. Western blots were performed as previously described withsome modifications. Briefly, cells were lysed with 100 μl of RIPA lysisbuffer with phosphatase and protease inhibitors (50 mM Tris HCl pH8, a50 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mMorthovanadate, 10 mM NaF, protease inhibitors (Sigma)). Proteinconcentration was measured to normalize protein loading using the BCAprotein assay. Adjusted amounts of protein was loaded and separated bySDS-PAGE (12%) and transferred onto PVDF membrane (Millipore, Mass.).Membranes were dried overnight and blocked for 1 hour with TBS-T+BSAfollowed by a 1-hour incubation with anti-phospho-ERK (Sigma) and signalwas detected using enhanced chemiluminescence (Amersham).

In Vivo Therapeutic Studies in Mice.

The murine NX3IT28 cell line was generated by hybridization of theGD2-negative C1300 murine neuroblastoma cell line (A/J background) withmurine dorsal root ganglional cells from C57BL/6J mice, as previouslydescribed. The NXS2 subline was then created by the selection of NX3IT28cells with high GD2 expression.

Six-to-seven-week-old female A/J mice, weighing 15-18 g were purchasedfrom Harlan Laboratories (Harlan Italy, S. Pietro al Natisone, Italy)and housed in sterile enclosures under specific virus and antigen-freeconditions. All procedures involving mice and their care were reviewedand approved by licensing and ethical committee of the National cancerResearch Institute, Genoa, Italy, and by the Italian Ministry of Health.A/J mice were pretreated with 1 mg/kg/day doses of human octreotide(OCT, ProSpec-Tany TechnoGene, Rehovot, Israel) for 4 days given slowlythrough the tail vein in a volume of 100 μl and then injectedintravenously with murine neuroblastoma NXS2 cell line (200,000/mouse),as previously described. After tumor cell injection, some groups ofanimals were starved for 48 hours and then i.v. treated with 80 mg/kg ofEtoposide Teva (Teva Pharma B.V., Mijdrecht, Holland), administered as asingle dose. Additional daily doses of OCT were administered for 4 daysafter chemotherapy. Control groups of mice without diet starvation andOCT treatment were also investigated.

Octreotide pre-treatment: 4 days 1 mg/kg/day

NXS2: 200,000/mouse on day 4

STS: from day 4 to day 6 (after tumor cell injection)

Etoposide: 80 mg/kg on day 7

Octreotide post-treatment: days 8-11

To determine toxicity and efficacy, mice were monitored routinely forweight loss and general behavior. The animals were killed by cervicaldislocation after being anesthetized with xilezine (Xilor 2%, Bio98 Srl,Milan, Italy) when they showed signs of poor health, such as abdominaldilatation, dehydration, or paraplegia. Survival time was used as themain criterion for determining the efficacy of each treatment.

The statistical significance of differential survival betweenexperimental groups of animals was determined by Kaplan-Meier curves andlog-rank (Peto) test by the use of StatDirect statistical software(CamCode, Ashwell, UK).

In some experiments, four-week-old female CD1 mice (Harlan), weighing18-20 g were used to evaluate stress resistance after 60-hour dietstarvation. These animals, i.v. injected with 110 mg/kg etoposide, weremonitored routinely for weight loss and general behavior. Survival timewas used as the main criterion for determining the differential stressresistance after short-term starvation.

In other experiments, four-week old female athymic (Nude-nu) mice(Harlan), weighing 20-22 gr were used to evaluate stress resistanceafter a 48-hour starvation. These animals were i.v.-injected with 100mg/Kg etoposide and then monitored routinely for weight loss and generalbehavior. Survival time was used as the main criterion for determiningthe resistance to high-dose chemotherapy after short-term starvation.

Thirty-four to forty-two-week old LID and their control L/L-mice wereused to evaluate the role of circulating liver-derived IGF-I in stressresistance. LID and L/L-mice were generated using the Cre/loxP system asdescribed before. Briefly, albumin-cre transgenic mice were crossed withmice homozygous for exon4 of the igf-1 gene flanked with two loxP sites(L/L), therefore generating mice deficient in producing hepatic IGF-I.L/L-mice have exon4 of the igf-1 gene flanked by loxP sites and producenormal hepatic IGF-I levels while LID mice show 75% lower circulatingIGF-I levels. All mice were individually caged throughout the experimentand had access to food and water ad libitum. Both LID and L/L-mice wereeach injected 500 mg/kg of cyclophosphamide i.p. using 29G insulinsyringes. Cyclophosphamide was prepared in saline at 40 mg/ml and warmedprior to injection. Mice were routinely monitored for weight loss andsigns of pain and stress. Survival time was the main criterion fordetermining the effect of low circulating IGF-I on the resistance tohigh-dose cyclophosphamide. The statistical significance of differentialsurvival between experimental groups was determined by Kaplan-Meiercurves and log-rank test using GraphPad Prism 4 software (San Diego,Calif.).

REFERENCES

-   1. Brachmann, C. B. et al. Designer deletion strains derived from    Saccharomyces cerevisiae S288C: a useful set of strains and plasmids    for PCR-mediated gene disruption and other applications. Yeast 14,    115-32 (1998).-   2. Morano, K. A. & Thiele, D. J. The Sch9 protein kinase regulates    Hsp90 chaperone complex signal transduction activity in vivo. Embo J    18, 5953-62 (1999).-   3. Fabrizio, P. et al. Sir2 blocks extreme life-span extension. Cell    123, 655-67 (2005).-   4. McCarthy, K. D. & de Vellis, J. Preparation of separate    astroglial and oligodendroglial cell cultures from rat cerebral    tissue. J Cell Biol 85, 890-902 (1980).-   5. Hansen, M. B., Nielsen, S. E. & Berg, K. Re-examination and    further development of a precise and rapid dye method for measuring    cell growth/cell kill. Journal of Immunological Methods 119, 203-10    (1989).-   6. Yung, H. W. & Tolkovsky, A. M. Erasure of kinase phosphorylation    in astrocytes during oxygen-glucose deprivation is controlled by ATP    levels and activation of phosphatases. J Neurochem 86, 1281-8    (2003).-   7. Greene, L. A. et al. Neuronal properties of hybrid neuroblastoma    X sympathetic ganglion cells. Proc Natl Acad Sci USA 72, 4923-7    (1975).-   8. Lode, H. N. et al. Targeted interleukin-2 therapy for spontaneous    neuroblastoma metastases to bone marrow. J Natl Cancer Inst 89,    1586-94 (1997).-   9. Yakar, S. et al. Normal growth and development in the absence of    hepatic insulin-like growth factor I. Proc Natl Acad Sci USA 96,    7324-9. (1999).-   10. Kempermann, G., Knoth, R., Gebicke-Haerter, P. J., Stolz, B. J.,    Volk, B. Cytochrome P450 in rat astrocytes in vivo and in vitro:    Intracellular localization and induction by phenyloin. J Neurosci    Res 39, 576-88 (1994).-   11. Geng, J., Strobel, H. W. Expression, induction and regulation of    the cytochrome P450 monooxygenase system in the rat glioma C6 cell    line. Brain Res 784, 276-83 (1998).

Example IV Oncogene Homologs and the Regulation of Stress Resistance andAging in Lower Eukaryotes

The studies in S. cerevisiae and those in worms, flies, and mice haveuncovered a strong association between life span extension andresistance to multiple stresses. A remarkable resistance to multiplestresses including chemotherapy drugs and oxidants in long-lived yeastcells lacking pro-growth proteins including the orthologs of the humanRas (RAS2) and Akt (SCH9/AKT) proto-oncogenes was observed (FIG. 15).Both Ras and Akt are among the signal transduction proteins mostfrequently found in a constitutively activated form in human cancers.Stress resistance is also associated with long-lived worms and mice withreduced activity of homologs of the IGF-I receptor (IGF-IR), whichfunctions upstream of Ras and Akt in mammalian cells. This resistance isalso observed in calorie restricted model systems in which the calorieintake is reduced by 30 to 100%.

The discovery of the role of Ras2 and Sch9/Akt in the negativeregulation of stress resistance together with the association betweenmutations that activate IGF-IR, Ras or Akt and many human cancersprompted the hypothesis that normal but not cancer cells would respondto starvation or down-regulation of growth hormone (GH)/IGF-I signalingby entering a chemotherapy resistance mode. In fact, one of the majorphenotypic characteristics of malignant cells is the ability to grow orremain in a growth mode even in the absence of growth factors, in partprovided by the hyper- or constitutive activation of pathways includingthose regulated by IGF-IR, Ras and Akt.

There are many similarities between long-lived mutants in S. cerevisiae,C. elegans, Drosophila, and mice and many of the genes implicated inlife span regulation are also frequently found in mutated oncogenicforms in cancer cells. C. elegans age-1 and daf-2 mutations extend thelife-span in adult organisms by 65 to 100% by decreasing AKT-1/AKT2signaling and by activating transcription factor DAF-16. These changesare associated with the induction of superoxide dismutase (MnSOD),catalase, and HSP70. In previous work it was showed that theinactivation of the Ras/cAMP/PKA pathway in S. cerevisiae increaseslongevity as well as resistance to oxidative and thermal stress in partby activating transcription factors Msn2 and Msn4, which induce theexpression of genes encoding for several heat shock proteins, catalase(CTT1), and the DNA damage inducible gene DDR2. MnSOD also appears to beregulated in a similar manner. Previous results also suggest that theeffects of sch9/akt deletion on stress resistance and life span requirethe serine threonine kinase Rim15 and transcription factor Gis1. Inworms, SMK-1 functions downstream of DAF-16, to regulate UV andoxidative stress but not thermal stress. Thus, the yeastRas/Cyr1/PKA/Msn2/4 and Sch9/Rim15/Gis1 and the C. elegansDAF-2/AGE-1/AKT/DAF16 pathways play similar roles in regulatinglongevity and multiple stress-resistance systems. Similarly, inDrosophila, mutations in the insulin receptor/Akt/PKB pathway extendlongevity and a mutation in the G-protein coupled receptor homolog MTHcauses a 35% life span extension and increases resistance to starvationand paraquat toxicity. Life span is also extended by potentialdownstream mediators of starvation-responses such as the Indy gene.

Whereas the inactivation of these partially conserved pro-aging pathwaysincreases resistance to oxidative stress in lower eukaryotes, theconstitutive activation of analogous pathways is known to promotecancer. One object is to test whether the inactivation of the Ras andSch9/Akt pathways in S. cerevisiae by either deletion mutations orstarvation increases resistance to a range of chemotherapy drugs,including alkylating agents, topoisomerase inhibitors, and oxidants.Furthermore, another object is to test whether the overexpression of theoncogene-like RAS2 or SCH9/AKT genes can reverse the resistance ofmutated or starved yeast to chemotherapy drugs. In addition to testingthe range and mechanisms of DSR in S. cerevisiae, another object is tocharacterize the DNA damage caused by different categories ofchemotherapy drugs and study the mechanisms responsible for the effectof Ras and Sch9/Akt on the resistance to chemotherapy. The understandingof the mechanisms of DSR in yeast is important because it provides thefoundation for the development of an analogous method that can beapplied to killing cancer but not normal mammalian cells.

Oncogene Homologs and the Regulation of Stress Resistance in Mammals

The inactivation of signal transduction pathways implicated in cancerhas also been shown to increase resistance to stress and survival inmammals. The deletion of the p66^(SHC) gene, associated with Ras,increases resistance to paraquat and hydrogen peroxide and extendssurvival by 30% in mice. Mutations that cause either a deficiency in thelevel of plasma growth hormone (GH) and IGF-I or a reduction of IGF-Isignaling cause an up to 50% increase in life span. As observed inlong-lived lower eukaryotes, the activities of antioxidant enzymessuperoxide dismutases and catalase are decreased in murine hepatocytesexposed to GH or IGF-I and in transgenic mice overexpressing GH. Inrats, IGF-I attenuates cellular stress response and the expression ofstress response proteins HSP72 and hemeoxygenase. IGF-I is a polypeptideinvolved in many aspects of growth and development. IGF-I is generatedmainly in the liver and is found in the blood in a stable complex withIGF-I binding proteins. Homozygous Ames dwarf mutations in the Prop-1gene (df/df) prevent the generation of the anterior pituitary cells thatproduce growth hormone (GH), thyroid stimulating hormone, and prolactin.df/df young adult mice are approximately one third of the size ofcontrol mice but survive >50% longer than controls. This effect of dwarfmutations on life span appears to be caused by the absence of plasma GH,which stimulates the secretion of IGF-I from liver cells by activatingthe hepatic GH receptors. In fact, IGF-I is reduced dramatically in theplasma of df/df mice. The plasma GH deficiency appears to mediate theeffects of Prop-1 and Pit-1 mutations on longevity, since the mice thatcannot release GH in response to growth hormone releasing hormone alsolive longer. Furthermore, dwarf mice with high plasma GH, but a 90%lower IGF-I (GHR/BP null mice) live longer than the wild type mice.Taken together these studies suggest that the reduction in plasma IGF-Iis responsible for a major portion of the life span increase in dwarf,GH deficient, and GHR/BP null mice.

A recent study shows that deletion of the IGF-I gene exclusively in theliver (IGF-I/loxP) causes a dramatic reduction of plasma IGF-I, but doesnot result in growth and development defects suggesting that GH canaffect cellular function and the autocrine/paracrine release of IGF-I invarious tissues independently of plasma IGF-I. In fact, IGF-I mRNAexpression is normal in fat cells, muscle, kidney, heart and spleen ofIGF-I/loxP mice. Mutations that decrease the activity of the adenylylcyclase/cAMP/PKA pathway and increase life span and stress resistancewere identified. Recently, a reduction in adenylyl cyclase activity bydeletion of the 5 adenylyl cyclase (AC5) gene was shown to extend lifespan and also increase resistance to oxidative stress in mice,suggesting that the role of both the Akt and cAMP/PKA pathways in theregulation of aging and stress resistance may be conserved from yeast tomice (FIG. 15). The mammalian IGF-1 receptor activates both Akt/PKB andRas and analogously to yeast Sch9/Akt and Ras, regulates glucosemetabolism and cellular proliferation. Many studies, implicate increasedIGF-I or IGF-I signaling as a risk factor in a variety of cancers,suggesting that this pro-mitotic pathway can promote both aging but alsothe conditions necessary for cancer. Another object is to test whetherreduced glucose, serum, and/or IGF-I can protect primary mammalian cellsbut not, or less, rodent and human cancer cells against differentchemotherapy drugs.

Caloric Restriction, Dwarf Mice, and IGF-I

The chronic reduction of calories by 10-40% below ad lib intake slowsaging and extends life span in lab rodents. CR decreases age-relatedpathologies including tumors and kidney degeneration, lowers bloodglucose, IGF-I, and insulin, and elevates corticosterone (Table 3). CRalso causes a reduction in oxidative modifications of long-livedmacromolecules such as collagen and affects age-related changes in thebrain: it slows the loss of dopamine receptors and attenuates thedegeneration of spinal motoneurons in rats. Although the mechanismresponsible for the effects of CR is unknown, it may involve a decreasein the levels of circulating Insulin-like Growth Factor 1 (IGF-I). Theresults in yeast indicate that the reduction of Ras-cAMP-PKA andSch9/Akt signaling plays an important role in the effect of calorierestriction/starvation on stress resistance and aging (FIG. 17). Infact, CR mice and dwarf mice, with IGF-I signaling deficiency, shareseveral characteristics (compared to ad lib controls):

TABLE 3 Similarities between caloric restricted and dwarf mice CR Amesdwarf Body weight (young) reduced reduced Plasma glucose reduced reducedPlasma Insulin reduced reduced Body temperature reduced reduced PlasmaIGF-I reduced absent Corticosterone elevated elevated Life span extendedextendedStarvation Response in Mammals

The DSR method is based on the ability of starvation to protect normalbut not, or less, cancer cells against high dose chemotherapy. However,the use of starvation to protect cancer patients that often have alreadylost much weight would have limited applications. Therefore, it isimportant to identify factors that can mimic the effect of starvation onprotection. In the results the effect of the somatostatin analogoctreotide in DSR were tested but found to be ineffective in theprotection against the chemotherapy drug etoposide but effective in theenhancement of the toxicity to cancer cells but not normal cells afterstarvation. In other words, octreotide did not protect mice againstetoposide, but reverse the effect of starvation in the protection ofcancer cells but not normal cells. The explanation for these results iscomplex. In fact, the level of somatostatin, which inhibits the releaseof growth hormone from the pituitary, was found to be reduced by 70%after a 3-day fasting period (rats) but a 24-72-hour starvation inhibitsGH secretion, although this effect may not depend on somatostatin. Inhumans, a 48-hour starvation raises GH levels and does not increaseIGF-I level. This effect may be the result of a major increase in theIGF-I inhibitory protein IGFPB-1, which may decrease IGF-Ibioavailability and consequently prevent the feedback inhibition of GHsecretion by IGF-1. However, a longer starvation (5 days) does decreasethe level of IGF-I in humans. Thus, the decrease in the pro-growth IGF-Iby long-term starvation may contribute to the effect of starvation inthe protection against high dose chemotherapy but somatostatinadministration alone is not sufficient for protection. Another object isto study further the effect of different periods of starvation on thelevel of IGF-I and IGF-I binding proteins in mice. These studies areimportant for identifying the factors and mechanisms that mediate theeffect of starvation on resistance to chemotherapy.

Self-Sufficiency in Growth Signals and Insensitivity to Anti-GrowthSignals in Cancer Cells

Two of the major “alterations in cell physiology that dictate malignantgrowth” are: 1) self-sufficiency in growth signal, 2) insensitivity toanti-growth signals. In fact, the great majority of normal cells areunable to grow in the absence of growth factors, whereas cancer cellsexpress oncogenes that mimic the effect of growth factors. Such“independence” can be caused by changes in extracellular factors thatpromote growth, the activation of membrane receptors, or mutations thatlead to the activation of intracellular signal transduction proteins.Some of the extracellular factors overproduced by cancer cells includeTGF, PDGF and IGF-I. Among the receptors overexpressed in a number ofcarcinomas are the EGF and HER-2 receptors. Extracellular matrixreceptors that can promote growth, known as integrins, are also found tobe activated in cancer cells. Perhaps the most important contribution tothe “growth factors independence of cancer cells” comes fromintracellular pathways, particularly the Ras/Raf/MAPK and thePTEN/PI3K/AKT pathways. Altered forms of Ras that can render the cellgrowth independent from extracellular growth signals are found inapproximately a quarter of all cancers and half of colon cancers (FIG.16). The link between Ras and PI3K may further stimulate growth and alsoactivate anti-apoptotic pathways.

Another highly relevant feature of cancer cells is the ability to“disobey” anti-growth orders. These orders are often focused on theblock of the progression through the G1 phase of the cell cycle. Theretinoblastoma protein (Rb) is one of the central negative regulators ofproliferation in normal cells. When bound to transcription factor E2F,Rb prevents the induction of many pro-growth genes. One of the majorregulators of Rb and the anti-proliferation mode is the signalingprotein TGF-beta, which can block the phosphorylation that causes theinactivation of Rb. Cancer cells can become insensitive to anti-growthsignals in many ways including the down-regulation of the TGF-betareceptor or through mutations that cause the inactivation of Rb.

The “self-sufficiency in growth signals and insensitivity to anti-growthsignals” features of cancer cells and the central roles of Ras and Aktin oncogenesis (FIG. 16) are central to the belief that normal but notcancer cells will enter a protective mode in response to starvation orto the inhibition of growth factor signaling and in particular IGF-I.Based on the conserved entry into a “stress resistance” mode in responseto starvation or anti-growth signals in model systems ranging from yeastto mice, it is believed that all normal cells can enter a state in whichthey are protected or partially protected against chemotherapy, althoughit is not yet known how effective this protection will be. By contrast,the “self-sufficient” cancer cells should disobey or partially disobeythe “stress resistance orders” and continue on their pro-growth path.

IGF and IGFBP Alterations in Animal Models of Cancer

Transgenic mice that develop prostate tumors show an IGF axisdisturbance very similar in their behavior to the human disease. It hasbeen shown that TRAMP mice have IGF-I and IGF-IR alterations similar tothose proposed in the human disease. Similar findings have beendemonstrated in the Myc-over-expressing mouse model. Additionally, ithas been demonstrated that various interventions that affect thedevelopment of cancer in mice act by modulating the IGF axis; includinggreen tea extract, that increases IGFBP-3 and lowers IGF-I levels; andthe flavanoids quercetin and apigenin. It was observed that IGFBP-3directly induces apoptosis in cancer cells and that it mediates celldeath induced by other agents, including p53, which mediates IGFBP-3actions. Several cytokines, including TGFβ and TNFα and anticancerdrugs, such as retinoic acid can also induce IGFBP expression as well asapoptosis. The action of these agents appear to require the expressionof IGFBP-3 for the induction of apoptosis, as their effects are blockedby reagents that specifically block IGFBP-3 (such as siRNA, antisenseoligomers, and neutralizing antibodies). IGFBP-3-induced apoptosis hasnow been detected in multiple models, and IGFBP-3 has been identified asa target of the HPV oncoprotein E7, that targets it to degradation,thereby preventing its apoptotic effects. It was also demonstrated thatIGFBP-3 is a downstream target of EWS/FLI-1 and is lost in sarcoma.IGFBP-3 was identified as a p53 target gene that was frequentlyinactivated by methylation in germ cell tumors and bladder carcinoma.IGFBP-3 inhibits IGF-I action on growth and survival of cancer and alsodirectly induces apoptosis in cancer cells. Another object is to testthe role of IGFBP3 in the resistance of mice to chemotherapy but also inthe sensitization of cancer cells to various chemotherapy drugs.

Pharmacological Blockade of the IGF Axis in Cancer

As a result of the overwhelming data dealing with the role of the IGFsystem in the development and progression of various cancers, over adozen pharmaceutical companies have initiated the development of IGFantagonists. Several classes of IGF blockers are now in clinical trials,including IGFBPs, IGF-receptor blocking antibodies, and various smallmolecule inhibitors. The most promising and in the most advanced stageof development are the IGF-I receptor antibodies which are in Phase IItrials in several types of malignancies and have shown promising effectsin early reports. Interestingly, in most published papers, IGF-IRblockade is synergistic with agents that block parallel pathways. Asdiscussed below, there is a clear rationale for combining IGF-IRblockade and chemotherapy, including likely synergistic efficacy as wellthe expected avoidance of host toxicity. Another object is to test therole of IGF-receptor blocking antibodies in the resistance of mice tochemotherapy but also in the sensitization of cancer cells to variouschemotherapy drugs.

Short-Term Starvation Induces Differential Stress Resistance AgainstOxidative Stress in Yeast

To test whether constitutively active oncogenes or oncogene homologs canprevent the switch to a protective maintenance mode in response tostarvation, whether acute starvation would be as effective in increasingstress resistance as it has been shown for long-term calorie restriction(CR) was first determined. Such long-term CR strategy would not beappropriate for chemotherapy treatments since it requires several monthsto be effective. DSR studies were first performed in S. cerevisiae. Ashort-term starvation paradigm, as well as the deletion of the SCH9/AKTand/or RAS2 genes, each of which mimics in part calorie restriction andwas shown to cause high resistance to oxidative stress, was selected. Itwas believed that the combination of these genetic manipulations withstarvation would maximize DSR. The combination of STS (switch fromglucose medium to water at day 1 [OD=9-10] and incubation in water for24-48 hours) with the deletion of SCH9/AKT or both SCH9/AKT and RAS2increased resistance to a 30-60 minute treatment with hydrogen peroxideor menadione a 1,000- to 10,000-fold compared to cells expressing theconstitutively active oncogene homolog RAS2^(val19) or cells lackingSCH9/AKT (sch9/aktΔ) but expressing RAS2^(val19) (SCh9/aktΔRAS2^(val19))(FIG. 17A). The rationale for this experiment was to model in a simplesystem the effect of the combination of short-term starvation and agenetic approach on the differential protection of normal and cancercells. The results show that the expression of the oncogene-likeRAS2^(val19) prevents the 1,000-10,000-fold protection caused by thecombination of STS and inhibition of Sch9/Akt activity.

The effect of another oncogene homolog (SCH9/AKT) on resistance tooxidants was also tested. As with RAS2^(val19), overexpression ofSCH9/AKT sensitized yeast cells to both H₂O₂ and menadione (FIG. 17B).Similarly to the effect of the deletion of RAS2 and SCH9/AKT, thedeletion of the homolog of TOR, another gene implicated in cancer,slightly increased the resistance to oxidants. Whereas the expression ofRAS2^(val19) completely reversed the protective effect of the deletionof SCH9/AKT, it only had a minor effect on the reversal of theprotective effect of the tor1Δ (FIG. 17B). This is an importantdifference because it suggests that it may be problematic to attempt toachieve DSR by inhibiting intracellular targets instead of inhibitingreceptors extracellularly since inhibition of certain cytosolic pathwaysmay also protect cancer cells in which the target is downstream of theoncogenic mutation. For example the inhibition of Akt may also protectcancer cells with a PTEN mutations that causes the constitutiveactivation of PI3K.

Short-Term Starvation Induces Differential Stress Resistance AgainstAlkylating Agents in Yeast

To test whether DSR would also occur after treatment of yeast with ahigh concentration of chemotherapy drugs, the effect of SCH9/AKTmutations on the toxicity caused by alkylating agents methylmethanesulfonate (MMS) and cyclophosphamide (CP, a widely usedchemotherapy drug) was studied. As a very simple model for the effect ofSTS and/or IGF-I inhibition on metastatic cancer mutants lackingSCH9/AKT were mixed in the same flask with mutants lacking SCH9/AKT butalso expressing RAS2^(val19) at a 25:1 ratio and the mixture was exposedto chronic treatment with CP or MMS. The monitoring of the viability ofthe two mixed populations was possible since each population can bedistinguished by growth on plates containing different selective media.Of the approximately 10 million sch9/aktΔRAS2^(val19) cells mixed with250 million sch9/aktΔ, less than 5% of the sch9/aktΔRAS2^(val19) cellssurvived a 48-hour treatment with 0.01% MMS whereas the great majorityof sch9/aktΔ survived this treatment (FIG. 17C). Similar results wereobtained with mixed sch9/aktΔRAS2^(val19)/sch9/aktΔ cultures treatedwith cyclophosphamide (FIG. 17D). An experiment in which each cell typewas treated with CP separately was also performed and a similardifferential stress resistance between cells expressing RAS2^(val19) andcells lacking SCH9/AKT was observed. These results in a very simplemodel system suggest that DSR has the potential to work effectivelyagainst metastatic cancer by selectively killing cancer cells but notnormal cells. Considering the very limited success of therapies aimed attreating metastasis, it is essential to explore further the potential ofnew strategies like DSR.

Short-term Starvation Induces Differential Stress Resistance AgainstCyclophosphamide in Mammalian Cells

To test the efficacy of the starvation-based DSR method on mammaliancells, primary rat mixed glial cells (astrocytes+5-10% microglia), threedifferent rat, one human glioma and one human neuroblastoma cell lineswere incubated in medium containing low serum and either normal (1 g/L)or low (0.5 g/L) glucose and then treated with the chemotherapy drug CP(1 g/L glucose is within the normal human blood glucose range whereas a0.5 g/L glucose concentration is reached in mammals during prolongedstarvation). The 1% serum concentration minimizes the contribution ofglucose from serum, which is approximately 1 g/L. To avoid majordifferences in proliferation, glia and glioma cells were allowed toreach a 100% confluency. Whereas 80% of glial cells were resistant to 12mg/ml CP in the presence of 0.5 g/L glucose, only 20% of the cellssurvived this treatment in 1 g/L glucose (FIG. 2A). The increased stressresistance at the lower concentration of glucose (0.5 g/L) was observedstarting at 6 mg/ml CP but became much more pronounced at 12 mg/ml CP(FIG. 2A). By contrast, the lower glucose concentration did not increasethe resistance of cancer cell lines including C6, A10-85, RG2 ratglioma, LN229 human glioma or human SH-SY5Y neuroblastoma cells to 12-14mg/ml CP (FIG. 2A). The lower glucose concentration actually decreasedthe resistance of CP to RG2 glioma cells to 6 and 8 mg/ml doses (FIG.2A). To determine whether the DSR is affected by the high cell densitythis experiment was also repeated with cells that were only 70%confluent and similar results were obtained.

To determine whether the constitutive activation of the Ras/Erk pathwaymay be implicated in the unresponsiveness of the glioma andneuroblastoma cells above to starvation the phosphorylation of Erk,which functions downstream of Ras, in 1% serum or serum-starved cellswas measured. The phosphorylation data indicate that 2 of the 5 linesmaintain a high level of Erk activity even after a 16-hour starvation(FIG. 11B), in agreement with the 30% frequency of Ras mutations inhuman cancers. The reduction of Erk phosphorylation but inability toincrease protection to cyclophosphamide in the other 3 lines (FIG. 2A)is consistent with an anti-resistance role of other common mutations inpro-growth pathways such as the PTEN/PI3K/AKT pathway. Notably, it isbelieved that the constitutive activation of any pro-growth pathway (notonly the Ras and Akt pathway) would make cancer cells unresponsive orless responsive to the starvation or IGF-I reduction-dependentprotection.

The experiments above were performed in medium containing 1.0 g/Lglucose and different concentrations of glucose. The effect of onlyreducing the level of serum from the standard 10% to 1% on the toxicityof high-dose cyclophosphamide was also tested. Treatment with 15 mg/mlCP was toxic to primary glial cells in 10% serum but the switch to 1%serum caused a reduction in toxicity (FIG. 2B). By contrast, the sameconcentration of CP was as toxic to C6 glioma cells in 10% serum as itwas in 1% serum (FIG. 2B).

In the S. cerevisiae experiments above it was showed that the deletionof SCH9/AKT protects whereas the constitutive activation of Ras2(RAS2^(val19)) or SCH9/AKT overexpression sensitizes the yeast cells tooxidants and/or alkylating agents. Since mammalian Ras and Akt are majorsignal transduction proteins downstream of the IGF-I receptor, andconsidering the role of the IGF-I pathway in regulating stressresistance, the effect of IGF-I and of an antibody against IGF-IR on DSRwas also tested. It was reasoned that primary cells would respond to theIGF-I-inhibiting treatment by increasing stress resistance whereascancer cells, which often express oncogenes that cause constitutive Rasand Akt activation, would not. Treatment with 100 ng/ml IGF-I (in thelow IGF-I range for human adults) caused a 3-fold increase in thetoxicity of cyclophosphamide to primary mixed glia but did not increasethe toxicity of CP to C6 glioma cells (FIG. 2C). Furthermore,pre-incubation with an anti-IGF-IR antibody (aIR3) protected primaryglia but not three glioma cell lines tested against CP toxicity (FIG.2D). A similar sensitizing role of IGF-I was observed with primary ratcortical neurons but not the rat pheochromocytoma tumor PC12 cell linetreated with oxidants. These results in normal glia and in rat and humanglioma and neuroblastoma cell lines are consistent with those in yeastcells and support the belief that short-term starvation and/or drugsthat down-regulate IGF-IR/Ras/Akt signaling can protect normal cellsmuch more effectively than cancer cells against chemotherapy. Notably,this differential stress resistance should apply to the great majorityof pro-growth oncogenic mutations and not only to cancer cells withmutations in the Ras, Akt or mTor pathways. In fact, it worked with allthe six cancer cell lines tested (FIG. 2), independently of cancerorigin.

Short-Term Starvation Induces Differential Stress Resistance AgainstChemotherapy in Mice

To test DSR in vivo mice were treated with high dose chemotherapy incombination with STS and/or GH/IGF-I lowering strategies. For thispurpose etoposide, a widely used chemotherapy drug which damages DNA bymultiple mechanisms and displays a generalized toxicity profile rangingfrom myelosuppression to liver and neurologic damage, was selected. A/Jmice were administered with a high dose of etoposide (80-110 mg/kg)after a period of starvation, GH/IGF-I lowering treatment or both. Inhumans, a third of this concentration of etoposide (30-45 mg/kg) isconsidered to be a high dose and therefore in the maximum allowablerange.

To reduce GH/IGF-I mice were pre-treated for four days with thesomatostatin analogue octreotide. This pre-treatment was followed byetoposide administration. A sub-group of mice were also starved for 48hours (STS) before treatment with etoposide. The mice pre-treated withoctreotide received this treatment for 4 additional days afterchemotherapy. Whereas 80 mg/kg etoposide killed 43% of control (Eto,n=23, 2 experiments) and 29% of octreotide pre-treated mice (Oct/Eto,n=17), none of the mice treated with octreotide and also pre-starved for48 hours died after 80 mg/kg etoposide treatment (Oct/STS/Eto/Oct, n=35)and only one of the mice that were only starved (STS/Eto, n=16) diedafter etoposide treatment (FIG. 7A). Remarkably, STS-octreotidepre-treated mice, which lost 20% of the weight during the 48 hours ofstarvation, regained all the weight in the four days after chemotherapy(FIG. 7B) whereas in the same period the control mice lost approximately20% of the weight (FIG. 7B). Control mice treated with etoposide showedsigns of toxicity including reduced mobility, ruffled hair and hunchedback posture whereas Oct/STS/Oct pre-treated mice showed no visiblesigns of stress or pain following etoposide treatment.

The effect of STS alone on the protection of mice of another geneticbackground (CD1) was also tested. To determine whether an extended STSstrategy can be effective against a higher concentration of chemotherapydrugs 110 mg/kg etoposide was administered and the starvation period wasalso increased to 60 hours. Based on the experiments with oxidativestress, it was determined that this period is the maximum STS thatprovides protection. Longer starvation periods can weaken the animalsand have the opposite effect. This concentration of etoposide killed allthe control mice (Eto 110) but none of the STS pre-treated mice (STS/Eto110, n=5) (FIG. 7C). As with the A/J mice, pre-starved CD1 mice lost 40%of the weight during the 60 hours of starvation but regained all theweight in the week after the etoposide treatment and showed no visiblesign of toxicity (FIG. 7D).

The effect of the STS-based method was similar in athymic (Nude-nu)mice, widely used in cancer research to allow the study of human tumors.Whereas 100 mg/kg etoposide killed 56% of the nude mice and all the miceco-treated with octreotide, none of the STS/Eto/Oct or STS/Eto treatedmice (48-hour starvation) died (FIG. 7E). As observed with the other twogenetic backgrounds, the pre-starved mice gained weight during theperiod in which the Eto-treated mice lost weight (FIG. 7F).

In summary, out of 70 mice from three genetic backgrounds that werestarved before etoposide treatment, only one mouse in the STS only groupand none of the mice in the STS/Oct group died (FIG. 7I). By contrast,out of the 63 mice treated with etoposide alone or etoposide andoctreotide, 34 died of toxicity. These results are consistent with theyeast and glia/glioma data showing increased resistance to chemotherapytoxicity in response to starvation. In mice, octreotide alone was notsufficient to protect against etoposide toxicity and virtually all theprotection was due instead to STS. The discrepancy between the effect ofIGF-I in vitro and octreotide in vivo may be due to the relativelymodest effect of octreotide on the reduction of circulating IGF-I level.

Transgenic Mice with Low Circulating IGF-1 (LID) Show IncreasedResistance Against Cyclophosphamide

To determine whether a much more severe reduction in IGF-I level thanthat achieved with octreotide can increase resistance to chemotherapy invivo as shown in vitro, the resistance against a high dose ofcyclophosphamide of male and female LID mice, in which the liver IGF-Igene was conditionally deleted, resulting in a 75-90% reduced serumIGF-I concentration, was studied. The LID mice treated with 500 mg/kgcyclophosphamide showed a remarkable improvement in resistance, with 30%mortality vs the 70% mortality for control mice (FIG. 7G, P<0.002).Furthermore, the LID mice lost an average of 10% of weight vs 20% weightloss in control mice (FIG. 7H). The 70% surviving LID mice also did notshow any signs of toxicity. Together with the in vitro results withIGF-I and anti-IGF-IR antibodies and the established role of starvationon the reduction of IGF-I levels, these data suggest that the inhibitionof IGF-IR signaling may mediate part of the effects of STS on resistanceto cyclophosphamide in vivo. Notably, IGF-IR antibodies have been usedsuccessfully in a number of studies to reduce the growth or increase thedeath of cancer cells and are currently being evaluated in clinicaltrials.

Short-Term Starvation in Combination with Octreotide Protects Mice butnot Cancer Cells Against High Dose Chemotherapy

To determine whether the differential stress resistance observed inyeast and mammalian cells would also occur in mice, the survival of miceinjected with cancer cells was followed. A particularly aggressiveneuroblastoma (NB) tumor line (NXS2) (NB is the most common extracranial solid tumor, and the first cause of lethality in pre-school agechildren) was selected. Advanced NB patients, who representapproximately 50% of the cases, show metastatic dissemination atdiagnosis, and have a long-term survival rate of only 20% in spite ofaggressive chemotherapy with autologous hematopoietic stem cell support.

The NXS2 neuroblastoma line in mice induces consistent and reproduciblemetastases in a pattern which resembles the clinical scenario observedin neuroblastoma patients at advanced stages of disease. Experimentalmetastases in the liver, kidneys, adrenal gland, and ovaries wereobserved after 25-30 days of the inoculation with 200,000 NXS2 cells(Table 2) as previously described. The tumor development and survival ofSTS/Eto treated mice was significantly different from that of controls(Gr. 7 vs. Gr. 1 p<0.0001) (FIG. 8A, Table 2) suggesting that STS aloneprovides strong protection to the mouse but only partial protection ofcancer cells against etoposide. Based on these results, the use of STSalone would require several or many chemotherapy cycles to obtaintoxicity to cancer cells comparable to that caused by high dosechemotherapy alone.

By contrast, none of the Oct/STS/Eto/Oct injected with NXS2 cells dieduntil day 46, at a point when all the controls had died of cancer (FIG.8A, Table 2) (Gr. 4 vs. Gr. 1 p<0.0001). One mouse from this groupsurvived until day 130. The survival of Oct/NXS2/STS/Eto/Oct mice wasnot significantly different from that of NXS2/Eto and Oct/NXS2/Eto/Octgroups (Table 2) but, as described above, 50% of mice not protected withSTS died of etoposide toxicity (FIG. 7A). Even if the initialetoposide-dependent deaths are not considered, the long-term survival ofthe octreotide/STS/NXS2/etoposide group was not significantly differentfrom that of the NXS2/etoposide. These results suggest that incombination with octreotide, STS protects the animal but not or muchless the cancer cells against chemotherapy. Although a single injectionof high close etoposide cannot be expected to cure the mice injectedwith NXS2 cancer cells, the high protection provided by STS/Oct againstthe initial chemotherapy toxicity to mice but not cancer cells providesa system that allows multiple or many cycles of etoposide treatment.Although the Eto alone group appears to perform slightly better than theSTS/Oct/Eto group, the initial high toxicity would prevent the use ofhigh dose Eto alone. Attempts to combine weekly injections of etoposidewith STS/Oct were discontinued because of the damage to the tails causedby the many i.v. injections, which prevented additional injections.Thus, the present results show that the differential stress resistancealso works in vivo.

Octreotide does not Sensitize NXS2 Neuroblastoma Cells to Etoposide

Somatostatin analogues have been reported to promote anti-tumor activitythrough two distinct effects: direct actions, mediated by somatostatinreceptors, and indirect actions, independent of the receptors. Thesomatostatin/octreotide receptor-mediated effect includes inhibition ofcell cycle and growth factor effects, and induction of apoptosis. Incontrast, the indirect effects comprise inhibition of the release ofgrowth factors such as growth hormone and IGF-I. To determine whetheroctreotide was acting directly on cancer cells the toxicity of etoposideto NXS2 cells cultured in vitro in the presence or absence of octreotidewas tested. Either 10 or 50 micromolar octreotide did not sensitize NXS2cells to etoposide treatment (FIG. 8B) suggesting that it is notincreasing the survival of the NXS2 injected mice by directlysensitizing the cells to etoposide. Because of the many studies showingan anti-tumor growth and survival effect of lower GH and IGF-I orinhibition of the IGF-I and GH receptors, these results suggest thatoctreotide may be improving long-term survival of STS treated mice byits well established role in decreasing GH and IGF-I levels althoughother effects cannot be ruled out. Notably octreotide and othersomatostatin analogues have been shown to have therapeutic effects in anumber of cancers. In the absence of short-term starvation, octreotidealone did not protect mice against NXS2-dependent death (FIG. 8A),suggesting that it is the synergism between etoposide and octreotidethat is effective in killing tumor cells. These results also suggestthat octreotide pre-treatment before the injection of NXS2 cells doesnot affect the tumor growth.

Targeted Deletion of Hepatic IGF1 in TRAMP Mice Leads to DramaticAlterations in the Circulating IGF Axis but does not Reduce TumorProgression

The role of systemic and local IGF-I in the development of prostatecancer is still controversial. TRAMP mice express the SV40 T-antigenunder the control of the probasin promoter, and spontaneously developprostate cancer. TRAMP mice was crossed with liver IGF-I deficient (LID)mice to produce LID-TRAMP mice, a mouse model of prostate cancer withlow serum IGF-I, to allow the study of the effect of circulatory IGF-Ilevels on the development of prostate cancer. LID mice have a targeteddeletion of the hepatic Igf1 gene but retain normal expression of Igf1in extra-hepatic tissues. Serum IGF-I and IGFBP-3 levels in LID andLID-TRAMP mice were measured using novel assays, which showed that theyare approximately 10% and 60% of control L/L-mice, respectively. SerumGH levels of LID-TRAMP mice were 3.5-fold elevated relative to L/L-TRAMPmice (p<0.001). Rates of survival, metastasis, and, the ratio ofgenitourinary tissue weight to body weight were not significantlydifferent between LID-TRAMP and L/L-TRAMP mice (FIG. 18). There was alsono difference in the pathological stage of the prostate cancer betweenthe two groups at 9-19 weeks of age. These results are in strikingcontrast to the published model of the GH-deficient lit/lit-TRAMP, whichhas smaller tumors and improved survival, and indicate that thereduction in systemic IGF-I is not sufficient to inhibit prostate cancertumor progression in the TRAMP model, which may require reduction of GHlevels as well. On the other hand, LID mice were less susceptible tochemotherapy toxicity as shown in FIG. 7G, indicating that in some andpossibly many cancers, the main benefit of IGF blockade, may in fact bein the potential for protection of normal but not tumor cells againstchemotherapy.

Development of Novel Mouse Specific IGF-Related Assays (IGFBP-3, IGF-I,and Other Mouse IGF Axis Analytes)

One object is to study the effects of GH/IGF lowering treatmentstogether with chemotherapeutic agents in mouse models. A key element instudying mice models involving IGF-related effects is the development ofmouse-specific IGF and IGFBP assays. Novel assays for the IGF axis havebeen developed. Highly accurate mouse-specific assays for mIGF-I,mIGF-II, mGH, mALS, mIGFBP-1, mIGFBP-2 and mIGFBP-3 have been developed(FIG. 19). These mouse-specific ELISA assays have been recentlydeveloped using rat-anti-mouse monoclonal antibodies and recombinantmouse IGF-related proteins from R&D Systems. These assays havesensitivities of less than 0.2 ng/mL, have no cross-reactivity withhuman homologues (human serum reads <1 ng/ml) and no interference withother IGFs or IGFBPs. Inter- and intra-assay CVs are <6% and recoveriesare 88-110% in the linear range of the assays. Importantly, a cleardevelopmental pattern for IGF-I, IGFBP-3, and ALS has been demonstrated,and it was showed that levels are low in GH deficient mice and in LID(Liver IGF-deficient) mice as well as being zero in the serum specificKO mice.

Starvation-Dependent Protection Against Chemotherapy

To understand whether STS is effective against a wide range ofchemotherapy drugs, its effect against the widely used drug doxorubicinwas also tested. A 48-hour STS effectively protected all mice (n=5)against 16 mg/Kg doxorubicin (FIG. 20). By contrast, the same dose ofdoxorubicin killed all the mice that were not pre-starved (FIG. 20).

Low IGF-1-Dependent Protection Against Chemotherapy

The role of IGF-I deficiency in the resistance to etoposide anddoxorubicin was tested. Surprisingly, the IGF-I deficient LID mice wereless resistant to etoposide compared to controls, although thedifference in survival was not significant (FIG. 21). By contrast, LIDmice were remarkably more resistant than controls to treatments withdoxorubicin. In this experiment multiple cycles of chemotherapy weremodeled by injecting the mice first with 20 mg/kg and 22 days later witha higher dose (28 mg/kg) of doxorubicin. All LID mice survived themultiple treatments whereas 75% of control mice died (FIG. 22).

Mechanisms of Regulation of Oxidative Stress and Chemotherapy Resistance

In the experiments shown below primary rat neurons were studied to beginto identify effectors of resistance to oxidative stress and chemotherapydownstream of Ras. It was showed that 2 inhibitors of MEK1/ERK,signaling kinases downstream of Ras, increase resistance to bothhydrogen peroxide and menadione (FIG. 23), providing evidence for a roleof ERK in regulating resistance to chemotherapy.

Starvation, Oncogene Homologs and Differential Resistance toChemotherapy Drugs in S. cerevisiae

The belief that the DSR system can protect normal but not cancer cellsagainst chemotherapy is based on the findings that starved yeast cellslacking oncogene homologs RAS2 or SCH9/AKT are resistant to oxidativestress as well as alkylating agents. In previous studies it was showedthat this resistance is reversed by the overexpression or constitutiveactivation of RAS2 or SCH9/AKT, which models oncogenic mutationscommonly found in human cancers. Thus, one object is to understandwhether and how starvation and/or down-regulation of these pathways canprotect normal cells but not cells expressing oncogene homologs againstdifferent classes of chemotherapy drugs. To dissect the mechanism ofprotection the role of various stress resistance transcription factorsinhibited by the Ras and Sch9/Akt pathways and in the resistance tothese chemotherapy drugs will be tested. Resistance to alkylatingagents, a topoisomerase II inhibitor, a thymidylate synthetaseinhibitor, and pro-oxidant drugs will be tested. Previous results pointto the error-prone polymerase Rev1 and recombination proteins in themediation of spontaneous age-dependent mutations. Here, the role ofthese error-prone enzymes and homologous recombination proteins in thesensitivity of cells expressing or overexpressing RAS and SCH9/AKT tochemotherapy agents will also be tested. Finally, a number of strainspreviously generated to characterize the range of DNA damage caused bythe various chemotherapy drugs and the effect of Ras and Sch9/Aktactivity on this damage will be used. Although results in S. cerevisiaedo not always reflect the biology of mammalian cells, the studiessuggest that oncogene homologs play conserved roles in the modulation ofresistance to chemotherapy drugs.

The following studies will be performed:

a) Determine the resistance of starved (48 hours in water) ornon-starved wild type, ras2Δ, sch9/aktΔ, ras2Δsch9Δ expressing eitherRAS2^(val19), SCH9/AKT or an empty vector to cyclophosphamide,etoposide, 5 FU, and menadione (vitamin K3). Both acute and chronictreatments will be performed (see FIG. 17 legend and detailed methods).Mix culture experiments as described in the legend of FIGS. 17C and Dwill also be performed. These chemotherapy drugs were chosen becausethey are commonly used to treat cancer (with the exception of menadione)but also because they represent different categories of toxicity: DNAalkylation (CP), topoisomerase II inhibition (ETO), thymidylatesynthetase inhibition (5FU), and oxidation-dependent single and doublestrand DNA breaks (menadione). RAS2^(val19) or SCH9 overexpression modelthe very common oncogene mutations that cause the activation of Akt orRas in cancer cells (see FIG. 17 legend and detailed methods).

b) Determine the role of the major stress resistance transcriptionfactors Msn2/Msn4, Gis1 and forkhead transcription factors Fhl1, Fkh1,Fkh2, and Hcm1 on the protection of sch9/aktΔ and ras2Δ mutants againstDNA alkylation, topoisomerase II inhibition, thymidylate synthetaseinhibition, and oxidation-dependent single and double strand DNA breaks.These transcription factors have been identified as major mediators ofresistance to multiple stresses in yeast, C. elegans, and mammaliancells. These studies will help identify the mediators of resistance tochemotherapy downstream of Sch9/akt and Ras and may point to humantranscription factors that play similar roles.

c) Study the mechanisms of RAS2- and SCH9/AKT-dependent sensitization ofcells against DNA damage in yeast cells treated with cyclophosphamide,etoposide, 5 FU, and menadione. Examine point mutations, smallinsertions/deletions, and gross chromosomal rearrangements using systemsdescribed below. Determine the role of Rev1 and Rad52 in the DNA damagecaused by the various chemotherapy agents by studying rev1Δ, rad52Δ,ras2Δ, sch9/aktΔ and sch9/aktΔrev1Δ, ras2Δrad52Δ, sch9Δ rad52Δ,ras2Δrev1Δ. Resistance to the chemotherapy agents in sch9/aktΔ and ras2Δmutants overexpressing REV1 or RAD52 will also be studied. Theseexperiments will test the role of error-prone polymerases and homologousrecombination in the toxicity caused by chemotherapy drugs.

One object is to confirm that the combination of starvation and the lackof both SCH9/AKT and RAS2 causes an up to 10,000-fold increase in theresistance to menadione and hydrogen peroxide but also caused a majorincrease in the resistance to alkylating agents cyclophosphamide andMMS. It is expected that resistance to these toxins will be reversed bythe overexpression of SCH9/AKT or by the constitutive activation of RAS2(RAS2^(val19)). However, the effect of either STS or ras2-sch9/aktmutations alone on DSR will be investigated further. In previous studiesit was also determined that 5FU and etoposide are toxic to yeast, asshow by others. It is believed that the DSR will also work with thesechemotherapy drugs.

Based on previous studies it is expected that stress resistance zincfinger transcription factors Msn2/Msn4 and Gis1 and to a lesser extendforkhead transcription factors (TF) Fhl1, Fkh1, Fkh2, and Hcm1 willmodulate protection against chemotherapy-dependent DNA damage. Althoughit has been shown that Msn2/Msn4 and Gis1 are effective against avariety of stresses, it cannot be excluded that different TF may provideprotection against different chemotherapy drugs.

It is also believed that the error-prone polymerase Rev1 plays a majorrole in the DNA damage caused by alkylating agents, oxidants andtopoisomerase II inhibitors as it did in the generation of spontaneousmutations which appear to be caused by a variety of insults includingoxidative damage. It will be very important to determine how each drugaffects DNA damage ranging from point mutations to gross chromosomalrearrangements (GCRs) both after acute and chronic treatments.

Another object is to investigate the role of error-prone polymerasessuch as Rev1 in the generation of chemotherapy-induced DNA damage inmammalian cells.

Starvation, IGF-I, Oncogenes and Differential Resistance to ChemotherapyDrugs in Primary Mammalian Cells and Cancer Cell Lines

The results indicate that reduction in the level of glucose or serumprotects primary glial cells more effectively than rodent or humanneuroblastoma and glioma cancer cells against high dosecyclophosphamide. It was also showed that the presence of IGF-Isensitized primary glia but not cancer cell lines againstcyclophosphamide. These results are consistent with the belief firsttested in S. cerevisiae, that Ras and Akt, which function downstream ofIGF-I in mammals, regulate stress resistance and that starvation orIGF-I receptor inhibition would differentially protect normal and cancercells against chemotherapy. Activation of the Ras or Akt pathway andpossibly of other pro-growth pathways by oncogenic mutations is expectedto force the cells into a pro-growth and low stress resistance mode. Oneobject is to perform experiments analogous to those for S. cerevisiae:the cyclophosphamide toxicity studies will be continued and extended todifferent toxic agents to begin to establish whether the STS/lowIGF-1-dependent DSR has the potential to be applied to a wide range ofchemotherapy treatments. Another object is to study the mechanismresponsible for the effect of oncogenes and proto-oncogenes onresistance to chemotherapy. The putative role of Ras and Akt insensitizing the cells to chemotherapy but also protecting againstapoptosis, at least under certain conditions will also be addressed.Finally, the mechanisms of protection with focus on Erk, p38, and FOXO3,which have been implicated as either positive or negative regulators ofstress resistance will be studied.

The following studies will be performed:

a) Investigate the role of low serum (1%) or low glucose (0.5 g/L) onthe resistance of primary glia and 7 lines of glioma and neuroblastomacells to different classes of chemotherapy drugs. Primary rat glialcells, rat glioma cell lines (C6, A10-85, RG2 and 9L), human glioma(LN229), mouse NXS2 neuroblastoma and human neuroblastoma (SH-SY5Y) celllines against cyclophosphamide, etoposide, 5 FU, and menadione will betested (see FIG. 2 legend and detailed methods). The focus on ratgliomas will enable one to determine the DSR between rat glia-derivedcancer cells and primary rat glial cells. In previous studies it wasshowed that the reduction of either serum from 10% to 1% or of glucosefrom 1 g/L to 0.5 g/L protected primary glia but not cancer cell linesagainst cyclophosphamide (FIG. 2). One object is to continue the studiesof the role of low serum or low glucose on the resistance tocyclophosphamide but also extend the studies to the different classes ofchemotherapy drugs listed above (see studies described above anddetailed methods). Both cell death (MTT, LDH assays) and apoptosis(Annexin V, TUNEL) will be measured.

b) Investigate the role of IGF-I in the resistance of primary glia andthe 7 lines of glioma and neuroblastoma to cyclophosphamide, etoposide,5 FU, and menadione. The effect of IGF-I and of IGF-I inhibitoryproteins including IGFBP-3 and IGF-I antibodies on stress resistance ofnormal and cancer cells will be studied (see FIGS. 2C and D and detailedmethods).

c) Investigate the effect of different concentrations of IGF-I, serum,and glucose on the activity of the Ras and Akt pathways in primary glialcells and cancer cell lines (FIG. 11B). The DSR method is based on thebelief that normal but not cancer cells will up-regulate stressresistance systems normally inhibited by the Ras and Akt pathways inresponse to starvation or a reduction in growth factors such as IGF-I.Because Ras and Akt are among the signal transduction proteins mostcommonly found in a constitutively active or up-regulated state incancers, whether either the Ras or Akt pathway or both remain activeafter the switch to low serum, low IGF-I, and low glucose as suggestedin the studies described above (FIG. 11B) will be determined. The 7different cancer lines listed above as well as primary rat glia will betested (see studies described above and detailed methods). For primaryglia the time course for the inactivation of the Ras and Akt pathwaysafter the switch to low serum, IGF-I, or glucose will also bedetermined.

d) Investigate the role of Ras and Akt in the regulation of resistanceagainst cyclophosphamide, etoposide, 5 FU, and menadione in primary gliaand determine whether cells are protected against necrosis or apoptosis(see detailed method). The yeast studies indicate that Sch9/Akt andpossibly Ras regulate oxidative stress-dependent DNA damage bycontrolling a pathway that includes serine/threonine kinases, and stressresistance transcription factors. One object is to test the role ofinhibition of Ras and Akt signaling in the protection against thechemotherapy drugs listed above in both primary glia and glioma cellslines. Whether the cell death is apoptotic or necrotic (see detailedmethod) will be determined. Another object is to study the role of Erk,p38, and FOXO transcription factors in the regulation of stressresistance downstream of Akt and Ras. These kinases and transcriptionfactors have been implicated in the regulation of stress resistance. Thephosphorylation status of Erk, p38, Akt, and FOXO3 in primary cellstreated with IGF-I (see detailed method) will be determined. Stillanother object is to test the effect of the inhibition of Erk, p38,PI3K/AKT activity and overexpression of FOXO3 transcription factors onthe resistance to chemotherapy drugs in primary rat glial cells treatedwith IGF-IR antibodies. Inhibitory drugs and/or siRNA will be used toreduce the activity of the above enzymes/transcription factors inprimary glia treated with IGF-I.

In the results described above (FIG. 2) starvation and/or reduction ofIGF-I was effective in protecting normal cells but not cancer cellsagainst the alkylating agents cyclophosphamide. Based on the results itis believed that the reduction in IGF-I signaling or starvation willalso protect against menadione and etoposide treatment (FIGS. 7 and 8B).As suggested by the studies described above (FIG. 11B) it is expectedthat Ras or Akt and downstream effectors will be constitutively activein the cancer cell lines but not in the primary glia. Thus, the removalor reduction of IGF-I, serum, or glucose should attenuate the activityof these pathways in glia but not glioma or neuroblastoma cells. The“apoptosis” experiments should enable one to discern the pro-damage andanti-apoptotic role of Ras and Akt. One possibility suggested by theexperiments described above is that the inhibition of Ras increasesapoptosis in the first 24 hours after treatment with oxidants, buteventually decreases necrosis. Based on the results described above, itis believed that Erk and to a lesser extent P38 mediate thesensitization to stress. This result is consistent with the opposingeffects of Erk and p38 in the regulation of apoptosis.

Another object is to understand how stress resistance affects otherprimary cells as well as cell lines representing additional types ofcancers including major ones such as breast and colon carcinomas. Yetanother object is to examine the role of additional signal transductionproteins and transcription factors in the resistance to chemotherapydrugs. For example, it would be useful to investigate additionaltranscription factors implicated in stress resistance such as FOXO1.

Starvation and Differential Resistance to Chemotherapy Drugs in Mice

The results in mice suggest that STS can be effective in the protectionagainst a dose of etoposide (topoisomerase II inhibitor) that killsapproximately 50% of the non-starved mice. It was also showed that STSalone only partially protects injected NXS2 neuroblastoma cells. Thisprotection was abolished by pre-treatment with the somatostatin analogoctreotide. One object is to test whether the combination of octreotideand STS is effective in the protection of mice against differentchemotherapy drugs (etoposide, 5 FU, and cyclophosphamide, andmenadione). Another object is to test the effect of STS/octreotideagainst several cancer cell lines using xenograft models (etoposideonly). In previous results only a single round of chemotherapy wasadministered, which was not sufficient to cure the mice from NXS2cancer. Because pre-starved mice did not show any visible sign oftoxicity, one object is to focus on multiple rounds ofSTS/octreotide/chemotherapy to attempt to kill all the injected cancercells or at least obtain a much greater delay of the cancer-dependentdeath in animals injected with cancer cells.

The following studies will be performed: In previous studies it wasshowed that STS protected against high dose etoposide. One object is totest whether STS is effective in the protection of mice against variouschemotherapy drugs. The role of STS on the protection of 4 differentmouse genetic backgrounds to high dose etoposide, 5 FU, andcyclophosphamide, and menadione will be studied. Different mouse strainswill be studied to allow xenografts with different types of cancer celllines and also to confirm that the STS strategy is effective in avariety of specific genetic backgrounds. The chemotherapy drugs abovewere chosen based on previous results and because they are among themost commonly used in the treatment of human cancers. The breast andovarian carcinomas models were added to test the effect of STS on morecommon carcinomas.

a) Test the effect of STS in combination with octreotide pre-treatmenton the resistance of C57BL6 mice against cyclophosphamide, etoposide, 5FU, and menadione.

b) Perform multiple injections of etoposide in combination with STS andoctreotide treatment in A/J mice pre-injected with NXS2 or Neuro2aneuroblastoma cells.

c) Perform multiple injections of etoposide in combination with STS andoctreotide treatment in athymic Nude (nu/nu) mice pre-injected withHTLA-230 or SH-5Y-SY human neuroblastoma cells.

d) Perform multiple injections of etoposide in combination with STS andoctreotide treatment in SCID mice pre-injected with MDA-MB1-231 humanbreast carcinoma.

e) Perform multiple injections of etoposide in combination with STS andoctreotide treatment in athymic Nude (nu/nu) mice pre-injected withOVCAR-3 human ovarian carcinoma.

f) To begin to understand the role of the GH/IGF-I axis in the DSReffect of STS, the effect of starvation on the level of growth hormone,IGF-I, IGFBP1, and IGFBP3 after the 48-hour starvation will be tested(see previous studies described above and detailed methods).

The systems listed above are among the major model systems for thetreatment of neuroblastoma. The ovarian carcinoma and breast carcinomasmodel systems were added to begin to understand whether theSTS/octreotide system is also effective in protecting normal but notother cancer cell types against high dose chemotherapy.

Based on previous results it is believed that STS will be consistentlyeffective against high dose etoposide and to a lesser extent againstcyclophosphamide. Octreotide is not expected to protect againstetoposide but may increase protection against other drugs and especiallymenadione. Because of the very low toxicity of etoposide in combinationwith STS and octreotide, the multiple injection experiments are likelyto be more effective against the injected NXS2, Neuro2A, HTLA-230,SH-5Y-SY, MDA-MB1-231, and OVCAR-3 cells compared to single injections.Based on previous results, octreotide should increase the toxicity toNXS2 cells and may increase the toxicity of etoposide against the cancercell lines listed above. In fact, it was showed that it had no directprotective effect on NXS2 cells (FIG. 8B) suggesting that it does notact through the activation of a specific receptor which may be specificto NXS2 cells. Naturally, octreotide may or may not reverse the smallprotective effect of STS on the other cancer lines as observed with NXS2cells. One problem was the damage to the tail of the mice caused by theinjections of cancer cell, chemotherapy drugs, and octreotide. To beginto address this problem mini-pumps for the slow delivery of octreotide(see detailed methods) has been already successfully used. Since 8injections of octreotide and one of cancer cells plus one forchemotherapy are performed in each cycle, the minipump will reduceinjections by 80%, which should allow many cycles ofSTS/octreotide/etoposide treatment.

Another object is to perform the multiple-injections experiment abovewith cyclophosphamide, menadione and 5-FU but eventually also with otherwidely used chemotherapy drugs such as doxorubicin. To begin tounderstand whether the STS method can be useful for human treatmentsimilar experiments with rats or other model systems will also beperformed.

Genetic and Pharmacologic Manipulations of the GH/IGF Axis in Mice andTheir Effects on Differential Resistance to Chemotherapy

A) One object is to utilize mouse models that have altered GH/IGF axis(including systemic IGF-deficient LID mice, local prostaticIGF-deficient PID mice, and the IGFBP-3 and IGFBP-1 KO mice) to decipherthe effects of local versus systemic IGF-1 and of IGFBPs on the host andcancer cells sensitivity to chemotherapy.

B) Another object is to determine the therapeutic index of chemotherapydrugs in these mice models that have been mated into a cancer (myc orTRAMP) background.

C) The third object is to investigate the role of IGFBP-3 and/orinhibitory antibodies against IGF-I either alone or in combination withSTS on the resistance of mice and of injected NXS2 neuroblastoma cellsto chemotherapy.

It is believed that 1) modulation of systemic (but not local), total orbio-available, IGF-I determines the differential toxicity of variouschemotherapeutic agents to mice and cancer cells; 2) higher doses ofvarious chemotherapeutic agents will be tolerated and will prove moreeffective in mice models of cancer with reduced total or bio-availablesystemic IGF-I.

In previous studies it was demonstrated that manipulations that decreasethe IGF-I signaling in a number of in vitro and in vivo models lead toincreased stress resistance in the host (or non-malignant) cells, butdid not affect and in some cases increased sensitivity to chemotherapyin the cancer or cancer-like cells. As discussed above, the circulatingIGF system is complex and IGF-signaling is controlled by both(endocrine, liver-derived) IGF-I and by locally produced(autocrine/paracrine) IGFs. Furthermore, IGF activity is regulated by anumber of IGFBPs, some of which (such as IGFBP-1) are themselvesup-regulated by factors such as starvation (inversely to IGF-I which isreduced by starvation and caloric restriction), while others, such asIGFBP-3 are regulated primarily by growth hormone and are down regulatedby somatostatin (in the same manner as IGF-I). A series of geneticallyaltered mice with a variety of specific modulations in the GH/IGF systemhave therefore been derived and/or acquired. These mice include: a)GHRKO mice with reduced growth hormone activity that harbor a GHreceptor deletion. These mice are small in size, have low systemic IGF-Iand low IGFBP-3 levels, and high IGFBP-1 levels. These mice havepreviously been shown to have delayed tumor development when mated intothe prostate cancer TRAMP model. b) LID (liver IGF-deficient) mice witha targeted deletion of the IGF-I gene in liver obtained. These mice arenearly normal in size, have very low systemic IGF-I levels, low IGFBP-3,and low IGFBP-1. As shown in previous studies, these mice displayreduced susceptibility to chemotherapy but do not have a reduced growthof prostate tumors when mated into the TRAMP model. c) PID (prostateIGF-deficient) mice with a prostate epithelial-specific deletion ofIGF-I, recently created by mating the prostate-specific probasin-cretransgenic mice with the Igf1-floxed mice. These mice are normal in sizeand have normal systemic IGF-I, IGFBP-3 and IGFBP-1. Their prostateIGF-I levels are currently being characterized. d) IGFBP3KO mice, whichhave been recently developed. These mice are 20% larger than controls,have undetectable IGFBP-3 levels, slightly reduced IGF-I, and normalIGFBP-1 levels in serum. e) IGFBP1KO mice. These mice are normal insize, have no IGFBP-1 and normal IGF-I and IGFBP-3. f) TRAMP mice, whodevelop early and aggressive prostate cancer as a result of prostatespecific expression of the SV40 T-antigen. It was recently showed thatthese mice respond to low fat diets by reducing tumor progression andhave elevated IGF-I levels. g) Myc mice, who develop slower growing andless aggressive prostate cancer as a result of prostate-specific overexpression of myc. It has been recently observed that these mice havehigher levels of IGF-I and also have a more aggressive tumor if fed ahigh fat diet. h) Myc mice have now been mated into the IGFBP-1 andIGFBP-3 knockout strains to create mice with cancer that lack each ofthe specific IGFBPs. It is believed that the IGFBP deletion willaccelerate the progression of the cancer and therefore chose a slowdeveloping tumor. The TRAMP model has been mated into the LID and PIDmodels with the expectation of delaying the progression of the tumors.

To determine the role systemic versus local IGF-I plays in vivo inchemotherapy resistance, LID mice will be compared to L/L-control miceas shown in previous studies. The PID mice will also be used as anadditional control. To determine the role of total versus bio-availableIGF-I IGFBP3KO and IGFBP1KO mice will be compared to control mice andtreated with chemotherapy inducing stress. To determine the role ofIGFBPs as mediators of the DSR effects of starvation and GH-blockadeIGFBP3KO and IGFBP1KO mice will be compared to control mice and treatedwith the 4 chemotherapy drugs as described above.

In each set of experiments there will be four groups of 20 mice pergroup mice: Group 1: WT mice treated with saline injections Group 2: WTmice treated with chemotherapy injections Group 3: Transgenic micetreated with saline injections Group 4: Transgenic mice treated withchemotherapy injections

As described above, one object is to use 4 groups of mice that will betreated with chemotherapy injections and to look at survival, serumIGF-related parameters and other markers of systemic stress and assessif any of the strains listed above are more or less resistant tochemotherapy. See above methods for details of treatments, which will besimilar to those in the immunodeficient mice.

To determine the role combinations and chemotherapy and IGF reduction orblockade in enhancing tumor suppression while improving survival thetreatments described above will be repeated on the genetic modelsdescribed herein. Prostate cancer is the most commonly diagnosed cancerin American men and a major health problem. While localized disease hasan excellent chance for cure, metastatic disease leads toandrogen-independent progression and death within a few years.Chemotherapy has been used clinically in prostate cancer with limitedsuccess (partially due to dose-limiting toxicity). Docetaxel is approvedin the US for hormone-refractory prostate cancer and represents animportant therapeutic milestone and is the current standard of care forthis disease. Cyclophosphamide as well as Etoposide have also beenexplored in human clinical trials, and while they are not universallyrecognized as beneficial in daily patient care, they do have effects athigh doses. Several prostate cancer bearing mice will be used as modelsfor testing the concept of differential stress resistance in response tochemotherapy. Docetaxel, etoposide, and cyclophoshamide will be tested.

There will be multiple sets of experiments conducted:

In each set of experiments there will be four groups of 20 mice pergroup mice: Group 1: Cancer Bearing WT mice treated with salineinjections Group 2: Cancer Bearing WT mice treated with chemotherapyinjections Group 3: Cancer Bearing Transgenic mice treated with salineinjections Group 4: Cancer Bearing Transgenic mice treated withchemotherapy injections

In each of the models of prostate cancer bearing transgenic mice theresponse to chemotherapy (docetaxel, etoposide, and cyclophoshamide)will be compared with the assumption that the combination of geneticablation of one of the components of the IGF system together withchemotherapy will enhance cancer treatment by protecting the host butnot cancer cells. Treatments will be given for 4 weeks at age 12 weeksfor TRAMP and 20 weeks for Myc.

Two primary outcome measures are: (1) prostate weight at sacrifice and(2) pathological grade of tumor. Secondary outcome measures willinclude: (a) TUNEL staining in prostate, (b) Ki67 staining, (c) serumIGF-I and IGFBP-3, (d) quantification of metastasis.

To investigate the role of inhibitory antibodies against IGF-I and/orIGFBP-3 either alone or in combination with STS on the resistance ofmice and of injected Nxs2 neuroblastoma cells to etoposide, menadione, 5FU, and cyclophoshamide (see detailed methods and FIG. 8). Theseexperiments are aimed at testing proteins/drugs with the potential toincrease protection against high dose chemotherapy. This is to establishwhether IGF-I and IGFBP3 are potential STS mimics. Studies with IGF-Iinhibitory antibodies and IGFBP3 have been carried out.

These experiments will be performed essentially as described above(FIGS. 7 and 8, and detailed methods). The IGF-I or IGFBP3 willessentially replace the octreotide/STS treatment.

As shown in previous data it is believed that LID mice and also theGHRKO mice will exhibit increased stress resistance. In addition, it isbelieved that both the IGFBP-1 and IGFBP-3 knock out mice will havereduced stress resistance. Furthermore, it is believed that IGFBP1KOmice will not show as much benefit from starvation, as the increase inIGFBP-1 expected is part of the IGF-limiting effects of the treatment.It is believed that PID mice will not have differential stressresistance, but they will have an increase in the response to chemo interms of tumor suppression. An increase in tumor sensitivity tochemotherapy is expected in the LID-TRAMP mice. In the BPKO-Myc modelsit larger tumors and a poor response to chemo is expected. In general,the treatment with IGF-I antibodies or IGFBP-3 should be consistent withthe results obtained with LID and GHRKO mice. Together the object is toestablish the complex interface between the GH-IGF system and theresistance of normal and cancer cells to chemotherapy using a series ofgenetic cancer models with specific IGF-related deletions.

Detailed Methods

Yeast Strains and Growth Conditions:

Experiments are carried out in wild type (DBY746 MATa, leu2-3, 112,his3D1, trp1-289, ura3-52, GAL⁺), and isogenic strains lacking eitherSch9 or both Sch9 and Ras2. Wild type (DBY746) and sch9Δ strainsexpressing hyperactive RAS2^(val19) were constructed by transformationwith a centromeric plasmid containing RAS2^(val19) (pRS406-RAS2^(val19),CEN URA3), and over-expressing REV1 or RAD52 were constructed bytransforming with multicopy 2μ plasmid carrying REV1 or RAD52 obtainedfrom Open Biosystems. Yeast strains lacking stress responsetranscription factors (Msn2, Msn4, or Gis1), forkhead transcriptionfactors (Fhl1, Fkh1, Fkh2, or Hcm1), REV1, and RAD52 were generated byone-step gene replacement. Yeast cultures are grown in liquid syntheticdextrose complete medium (SDC) with 2% glucose, supplemented with aminoacids, adenine, as well as a four-fold excess of tryptophan, leucine,histidine, uracil. Strains harboring the centromeric plasmid containingRAS2^(val19) were always grown in the absence of uracil to maintainselection.

Yeast Viability:

Overnight cultures are diluted to OD₆₀₀ 0.1 into SDC medium. After 24hours (day 1), the appropriate strains will be mixed 1:1 based on OD₆₀₀and incubated for 2 hours. The mixed cultures will be then treated witheither etoposide (300 μM), menadione (200 μM), and 5-FU (150 μM) inmedium or water (STS). Etoposide, menadione and 5-FU will be introduceddirectly into the mixed culture to a final concentration of 0.01%. Fortreatments in water (STS), mixed cultures will be centrifuged for 5minutes at 2,500 rpm and the media replaced with eitherdistilled/sterile water or drug-dissolved water. Viability will bemeasured by quantifying colony-forming units (CFUs) every 24 hours byplating onto appropriate selective media. Viability of individualstrains will be measured using the same method as for the mixedcultures.

DSR Assays in Yeast:

Stress resistance against etoposide, 5-FU, and menadione will bemeasured by spotting serial dilutions of control and treated cells aspreviously described. Briefly, 24 hours after the initial inoculation(OD=0.1) in SDC medium, cultures will be washed, resuspended andincubated in water for 48 hours with shaking. At day 3, cells will betreated with etoposide, 5-FU, or menadione for 60 min. Serial dilution(up to 1,000-fold) of the treated cultures were spotted onto YPD platesand incubated for 2-3 days at 30° C.

Mutation Studies in Yeast:

Mutation studies in yeast will be performed as previously described.Point mutations, frame shifts and gross chromosomal rearrangements willbe measured.

The Role of STS and IGF-1 Lowering Treatments in Protection AgainstChemotherapy In Vitro:

Cells described in previous studies will be used. Glucose and serumrestriction will be done as described before. IGF-I treatment will beperformed by incubating cells for 48 hours in DMEM/F12 with 1% FBS andrhIGF-I (100 ng/ml, ProSpec-Tany TechnoGene, Rehovot, Israel), which isshown to be within the IGF-I level range for middle age humans. Toantagonize IGF-1 receptor activity, cells will be incubated with theneutralizing monoclonal anti-IGF-1R antibody (1 μg/ml, Calbiochem) inDMEM/F12 1% FBS for 24 hours. To inhibit IGF-1 activity, cells will betreated with IGFBP-3 (200 ng/ml, Abcam) in DMEM/F12, 1% FBS for 24hours. Following STS/IGF-1 treatments, cells will be incubated withvarying concentrations of cyclophosphamide (6-15 mg/ml) for 10 hours,etoposide and 5-fluorouracil (0-120 μM) for 48 hours, and menadione(0-150 μM) for 24 hours in DMEM/F12 with 1% FBS. Cytotoxicity will bemeasured by the ability to reduce (MTT) as described in previousstudies. Apoptosis will be measured by Annexin V binding using theApopNexin™ apoptosis kit (Upstate) and DNA fragmentation using the TUNELapoptosis detection kit (Upstate) following manufacturer's instructions.

The Effect of STS on IGF-1R Signaling In Vitro:

Cells described in previous studies will be STS pre-treated as describedabove and Ras and Akt activity will be measured. Cells will be lysed atvarious time points of STS treatments and lysates will be tested for Rasand Akt activity using the Ras activity assay kit (Upstate) and the Aktwestern blot assay kit (Calbiochem) following manufacturer'sinstructions.

The Role of IGF-1R Signaling in DSR In Vitro:

Prior to chemotherapeutic drug treatments, cells will be pretreated toinhibit the activity of proteins downstream of IGF-1R including RAS,Akt, ERK, p38 and FOXO3a. To inhibit K, H and N-Ras isoforms, cells willbe treated with the farnesyl transferase inhibitor FTI 277 (10 uM) andthe geranyl transferase inhibitor GGTI-298 for 48 hours as previouslydescribed. To inhibit Akt signaling, cells will be treated withWortmannin (100 nM) and LY294002 (20 uM) for 1 hour. Erk will beinhibited using 10 μM of ERK inhibitors U0126 or SL327 and also PD98059and p38 kinases by SB203580 for 2 hours. siRNA will be used tospecifically down-regulate the FOXO3a transcription factor. In order todetermine the phosphorylation status of p38 and FOXO3a, cells will belysed following treatment with IGF-I as described. The lysates will besubjected to a Western blot analysis. Anti-phospho-p38 specific(Thr180/Tyr182) antibody and anti-p38 antibody will be used to detectp38 phosphorylation and total p38 respectively (Cell signaling). Antiphospho FOXO3a (Thr 32) and anti FOXO3a (Ser 253) will be used to detectFOXO phosphorylation. Anti-FOXO3a will be used to detect total FOXO3a(Abeam). Anti-phospho-Akt (Ser 253), anti-phospho-Akt (Thr 308) andanti-Akt will be used to detect phosphorylated and total Akt. FOXO3aover expression will be done using the pcDNA3 plasmid containing a 2,000bp FOXO3a insert (Invitrogen). 0.2 ug of the plasmid will be used totransfect 80,000 cells using Fugene (Roche) according to manufacturer'sprotocol. siRNA will be used to specifically silence the FOXO3atranscription factor. Briefly, 80,000 cells will be transfected with 0.2ug of the pSilencer (Ambion) plasmid that express siRNA oligos targetingFOXO3a using Fugene. All drugs will be from Sigma.

Chemotherapy-Induced Toxicity Measurements in Mice:

For all the experiments, the animals will be housed in sterileenclosures under specific virus and antigen-free conditions. Procedureswill be reviewed and approved by the Institutional Animal Care and UseCommittee (IACUC) of University of Southern California and UCLA, and bythe licensing or by the ethics committee of the National ResearchCenter, and by the Italian Ministry of Health. Major toxic side effectsof chemotherapy such as myelosuppression, gastrointestinal damage, DNAdamage, cell death and weight loss will be examined as an indicator oftoxicity/morbidity. Also mice will be monitored twice and weighed oncedaily for any signs of stress or pain and will be euthanized usinginhalant isoflurane (1-4%) followed by cervical dislocation if found so.At 60 hours after chemodrug administration, mice will receive a singlei.p. injection of BrdU (1 mg in 300 μl of PBS). At 72 hourspost-chemotherapy, mice will be deeply anesthetized by a singleintraperitoneal injection of 50 mg/kg Nembutal. Following anesthesia, awhole-body perfusion will be performed and blood will be collected viacardiac puncture through the left ventricle and stored in tubes withK3-EDTA for further analysis. Cervical dislocation will be performed toensure euthanasia and organs will be collected and fixed immediately.

Myelosuppression will be measured by a complete blood cell count with 24parameters using the HEMAVET® 950 FS hematology analyzer. 24 and 48hours after chemodrug injection, 20 μl of blood from the tail vein willbe collected and also from the heart at the time of sacrifice and storedin K3-EDTA containing tubes and immediately analyzed. Histologicalexamination of bone marrow from pelvic bones and spleen (a site ofextra-medullary hematopoiesis in mice) will also be examined forcellularity and content. Gastrointestinal and organ damage will beassessed by looking for areas of ulceration by macroscopic examinationand by histological evaluation of the integrity of the mucosa, areas ofinflammation, and by measuring the villus to glandular height ratio inthe small intestine. Also in pilot experiments, liver, kidney, lung,spleen and heart will be examined for confirmation of organ toxicity byH&E staining focusing on maintenance of architecture, areas of fibrosis.Also organ weight will be recorded. Only one organ most damaged will beselected for each individual drug, and will be examined along with thesmall intestine in full scale experiments. Organs will be collectedafter whole-body perfusion with buffered formalin under deep anesthesiawith Nembutal. Collected organs will be fixed with 10% formalin for 48hours and stored in 70% ethanol to be sent to the Norris Cancer Centerhistology core facility for histological preparations. Specimens will beparaffin embedded and sectioned to 4 μm thickness. DNA damage will bemeasured by BrdU incorporation as previously described with somemodifications. Briefly, 12 hours prior to sacrifice, mice will be i.p.injected with 1 mg BrdU dissolved in 300 μl PBS. 6 mice from each groupwill be randomly selected and BrdU incorporation will be measured fromliver and small intestine tissue samples with antibodies against BrdUfollowing manufacturer's instructions (Abeam). Histological sections ofthe small intestine and liver will be performed as described above bythe Norris Cancer Center histology core facility. Cell death will bedetermined by measuring blood LDH levels using QuantiChrom™ LactateDehydrogenase kit (BioAssay Systems, CA). Procedures will followmanufacturer's protocol.

Short-Term Starvation-Based Chemotherapy Resistance in Mice:

Mice from 4 different genetic backgrounds will be tested with 4different chemodrugs. Mice will be starved (48 hr) as described inprevious studies before prior to chemodrug administration. C57BL/6, A/J,athymic Nude, and SCID mice will be tested with cyclophosphamide,etoposide, 5-fluorouracil, and menadione. Pilot experiments will firstbe conducted to determine the optimum dosage of each drug in each mousestrain. 3 doses will be tested with 6 mice per dose. Once the optimumdosage is known, the procedures will follow as described in preliminarystudies with 25 mice per group. The number of mice were determined fromprevious experience and statistical calculations expecting, with 95%confidence (α=0.05, β=0.2), a 20% difference between the control andexperimental groups aided by Statsoft and SigmaStat.

The Role of the GH/IGF-1 Axis in Protection Against Chemotherapy inMice:

Antibodies against the IGF-1 receptor and IGFBP-3 will be employed. Thiswill be tested in at least two mouse strains with 4 drugs mentionedabove. Based on previous experience, a single injection of antibodieswill be given i.p. 2 days prior to chemodrug administration at 300μg/kg. As for IGFBP-3, daily i.p. injections with 4 mg/kg/day will begiven for 4 days prior to chemotherapy drug administration. 25 mice willbe employed per group as calculated above. The groups will be testedwith one chemotherapy drug at a time and the pre-treatments will be asfollows: Group 1: Control; Group 2: STS; Group 3: IGF-IR antibody; Group4: IGFBP-3; Group 5: IGF-1R antibody+IGFBP-3; Group 6: STS+IGF-IRantibody+IGFBP-3.

Liver IGF-1 Deletion (LID) Mice:

Due to the limited number of LID mice, 2 drugs will be first tested:5-fluorouracil and menadione. 1) cyclophosphamide: 18 LL- and 19 LIDmice will be given 300 mg/kg CP by a single i.v. injection. 2)Menadione: 11 LL- and 10 LID mice will be given 100 mg/kg menadione by asingle i.v. injection.

TRAMP and Myc and PID (Prostate Cancer Models):

3 drugs will first be tested: docetaxal (10 mg/kd per day, single i.p.injection); Etoposide Teva (Teva Pharma B.V., Mijdrecht, Holland) willbe injected 80-100 mg/kg; cyclophopshamide (Sigma) will be injected at300 mg/kg. See below and FIG. 7G for experimental details.

Growth Hormone Receptor Knock-Out (GHRKO) Mice:

4 heterozygous GHRKO breeding pairs will be received, as homozygous donot breed well. The average litter size is about 6 pups and the averageoffspring that is a homozygous GHRKO is 12%. Etoposide,cyclophosphamide, 5-fluorouracil and menadione will be tested, and inorder to obtain statistically significant results at least 20 mice pergroup is required as calculated above. Therefore, 8 groups includingcorresponding control groups (wild type GHR) are required, whichtranslates into 80 GHRKO and 80 control mice. The wild type controlgroup is in the inbred C57BL/6 background. The mating scheme will be asfollows. By mating only heterozygotes, it is estimated to take 72 weeksto obtain the desired number of mice. Mice will be genotyped at the ageof 6 weeks with DNA obtained by tail biopsies following JacksonLaboratory protocols. Since GHRKO mice are in the C57BL/6 background,optimum drug dosages would be derived from previous experimentsmentioned above.

Starvation-Based Differential Chemotherapy Resistance in Mice:

The following cancer models will be performed: Murine experimentalmetastatic neuroblastoma model in syngeneic A/J mice: The murine NX31T28cell line was generated by hybridization of the GD2-negative C1300murine neuroblastoma cell line (AIJ background) with murine dorsal rootganglional cells from C57BL/6J mice, as previously described. The NXS2subline was then created by the selection of NX3IT28 cells with high GD2expression. Six-to-seven-week-old female A/J mice, weighing 15-18 g(Harlan Italy, S. Pietro al Natisone, Italy) will be intravenouslyinoculated with 200,000 NXS2 cells. Intravenous injection of NXS2results in experimental metastases to distant organ sites, includingliver, kidneys, ovaries, adrenal gland and bone marrow, as previouslydescribed (Lode N H et al, JNCI 1997). Murine thoracic neuroblastomamodel in syngeneic A/J mice: Six-to-seven-week-old female A/J mice(Harlan Laboratories), weighing 15-18 g, will be injected in themediastinum, through the skin of the precardial area, with 1×10⁶ Neuro2acells, which have the same genetic background than NXS2. Precisely, theneedle will penetrate 3 mm and the syringe made an angle of about 120°with respect to the mediastinum. The pericardial area is located betweenthe second and the third rib. Fifteen days after tumor inoculum,numerous subpleural metastases can be detected in the lung and in thedraining lymphonodes, as previously described. Human experimentalpseudometastatic neuroblastoma model in Nude mice: Six-to-seven-week-oldfemale athymic Nude mice (nu/nu Harlan Laboratories), weighing 18-24 gwill be intravenously injected with 3×10⁶ human neuroblastoma HTLA-230cell line. This experimental model is characterized by the presence ofmacrometastases in different organs such as liver, kidneys, ovaries andadrenal glands, and micrometastases in the bone marrow, as previouslydescribed. Human orthotopic neuroblastoma model in SCID mice:Six-to-seven-week-old female SCID mice (Harlan Laboratories) will beanesthetized with ketamine (Imalgene 1000, Merial Italia s.p.a, Milan,Italy), subjected to laparotomy and injected with 1.5×10⁶ HTLA-230, inthe capsule of the left adrenal gland, as previously described. Humanmetastatic breast carcinoma model in SCID mice: Five-to-six-week-oldfemale SCID mice will be anesthetized with ketamine, and injected with1×10⁶ MDA-MB-321 human breast carcinoma cells in the mammary fad pad.The implantation of this tumor cell line induces the development of lungand lymph nodes metastases. Human intraperitoneal ovarian carcinomamodel in Nude mice: Five-to-six-week-old female athimic Nude mice willbe intraperitoneally injected with 1×10⁶ OVCAR-3 human carcinoma cellline as previously described. In all the models above described, thetherapeutic effect of different chemotherapeutic agents includingetoposide, cyclophosphamide, 5-fluorouracil (5-FU), and menadione willbe tested. All injections will be performed intravenously. Specifically,etoposide Teva (Teva Pharma B.V., Mijdrecht, Holland) will be injected80-100 mg/kg; cyclophopshamide (Sigma) will be injected at 300 mg/kg;5-FU will be injected at 150 mg/kg; Menadione will be injected at 50-100mg/kg. These agents, administered as a single dose or as multiple doses,will be used in combination with short term starvation (STS) andoctreotide (OCT). According to the protocol used for previousexperiments, the animals will be inoculated with tumor cells, starvedfor 48 hours and then intravenously treated with the chemotherapeuticagents. Additional daily doses of OCT will be administered for 4 daysafter chemotherapy. In some experiments, the cycle ofSTS-chemotherapy-OCT will be repeated after 1 week. Control groups ofmice without diet starvation and OCT treatment will be alsoinvestigated. For each cancer model, we will perform an experiment thatincludes 8 groups of animals (10 mice/group), as follows: 1) Controlgroup: 10 mice inoculated with tumor cells on day 1. 2) OCT: 10 miceinoculated with tumor cells on day 1 followed by OCT (1 mg/Kg: dailydose) administration for 4 days from day 4 to day 7. 3) STS: 10 miceinoculated with tumor cells followed by 48 hours-STS from days 1 to day3. 4) STS/OCT: 10 mice inoculated with tumor cells followed by 48hours-STS+OCT administration for 4 days. 5) Chemotherapy: 10 miceinoculated with tumor cells, followed by chemotherapy treatment on day3. 6) Chemotherapy/OCT: 10 mice inoculated with tumor cells followed bychemotherapy treatment/OCT administration for 4 days. 7)STS/Chemotherapy: 10 mice inoculated with tumor cells followed bySTS/chemotherapy treatment. 8) STS/Chemotherapy/OCT: 10 mice inoculatedwith tumor cells followed by STS/chemotherapy treatment/OCTadministration for 4 days. In additional experiments, the chemotherapyand OCT injections will be repeated every 2 weeks.

Octreotide Delivery Using Osmotic Pumps:

Mini-osmotic pumps (Alzet, Model 2004) will be implanted subcutaneouslyto instill octreotide over a 3 month period. Octreotide will bedelivered at 0.25 microl/hr at a concentration of 50 microg/kg/hour.Each mini-pump will deliver for 4 weeks and therefore will be replacedevery 4 weeks. Procedures will be performed as described by themanufacturer.

In Vivo Bioluminescence Labeling of Cancer Cells:

In order to study differential resistance to chemotherapy in vivo, A/Jmice will be intravenously injected with 2×10⁵ mouse neuroblastoma NXS2cell line stably expressing the firefly luciferase gene. Plasmidsexpressing the firefly luciferase gene were obtained as a SalI/BglIIfragment form pGL3-control vector (Promega), and cloned into theXhoI/BamHI sites of the retroviral pLXIN bicistronic vector. Monitoringthe growth and death of cancer cells in the animal will be possible bydetecting bioluminescence. This is a non-invasive method to monitor thegrowth and death of cancer cells at multiple stages of the experiment.25 mice will be employed in each group and 5 mice will be randomlyselected for bioluminescence imaging. Toxicity will also be measured aspreviously described. Bioluminescence will be monitored as previouslydescribed. Briefly, mice will be given a single i.p. injection ofketamine (50 mg/kg) and xylazine (10 mg/kg) followed by an i.v.injection of luciferin (50 mg/kg). 4.5 minutes later, when the luciferinis well distributed, mice will be examined with an IVIS 200 opticalimaging system (Xenogen Corp.). Signal intensity will be quantified asphoton count rate per unit body area per unit solid angle subtended bythe detector (units of photon/s/cm²/steridian). 3-dimentional images ofbioluminescence will be generated by the single-view diffuse tomographycapability of the IVIS 200 and LIVING IMAGE 3D v 2.50. Once injectedcancer cells have metastasized, mice will be starved for 48 hours incombination with IGF-1R antibodies or IGFBP-3 as described above priorto chemotherapy. Each drug (cyclophosphamide, etoposide, 5-fluorouraciland menadione) will be tested with 6 pretreatment schedules describedabove in the role of GH/IGF-1 axis in resistance to chemotherapyexperiment. Toxicity will be measured daily as described above for thefirst 3 days after chemotherapy, followed by monthly tests for 6 months.

IGF-Related Hormone Levels in Mouse Serum:

Novel mouse assays for the IGF axis have been developed or recentlypublished. ELISA for mouse IGF-I, BP-1, 2, 3 and ALS and GH: Blood willbe collected at the time of sacrifice from an intra-cardiac source orthe retro-orbital sinus of the mice. Novel IGF-related mouse assays havebeen recently pioneered. mIGF-I, mIGFBP-3, IGFBP-1, IGFBP-2, and mGH andmALS levels will be run when appropriate. Prostate cancer mice mating:Colonies of Myc and TRAMP mice are currently in-house in UCLA breedingfacility. At 4 wk of age, pups will be weaned and sexed and tail snipsfrom all healthy offspring used for genotyping as required. PCR-basedgenotyping for mIGFBP3, mIGFBP1, mIGF1, Alb-cre and probasin-cretransgenics, and the T-antigen are routinely employed.

Clinical Examination:

Animals will be observed for mortality/moribundity twice daily duringthe week and once daily on weekends and holidays. Body weights andclinical observations all animals will be recorded weekly. Clinicalobservations will be made at 7 wk of age, 1 wk prior to treatment, priorto treatment on d 1, and weekly thereafter.

Preparation and Analysis of Prostate Tissues:

At the time of sacrifice, the lower GU tract, including the bladder,testes, seminal vesicles, and prostate, will be removed en bloc. The GUwet weight will be recorded to the nearest 0.01 g. Tissues collected atnecropsy will be fixed in 10% (Vol:Vol) phosphate-buffered formalin for12 h and then transferred to 70% ethanol before standard tissueprocessing (except for a sample that will be used for RNA). Sections ofthe prostate (4 mm) will be cut from paraffin-embedded tissues andmounted on ProbeOn-Plus slides (Fisher Scientific). Distant sitemetastases will be examined at the time of necropsy and dissected ifidentified.

Assessment of Prostate Histology and Immunohistochemistry:

Prostate and metastatic tumor histopathology will be determined by H&Estaining, and prostate immunohistochemical analysis of phospho-IGF-IR,phospho-Akt, COX-2, TUNEL will be quantified as described. Thetechniques for immunostaining and analyzing the stained tissue (percentof cells staining positive, intensity of staining, location of staining)will be used as described.

Image Analysis:

Sections will be visualized on a Zeiss-Axiophot DM HT microscope. Imageswill be captured with an attached camera linked to a computer. Imagesand figures will be composed by using ADOBE PHOTOSHOP 5.5 (AdobeSystems, Mountain View, Calif.). The initial section will behematoxylin-and-eosin (H&E)-stained; 10 unstained sections will follow.Subsequently, at 200 μm deeper into the block, another H&E will befollowed by 10 unstained sections. Histopathological evaluation usinglight microscopy will be performed on all H&E-stained slides preparedfrom the prostate of each animal.

Immunofluorescence Analysis and Apoptosis Detection:

Four micrometer-thick sections will be cut from paraffin-embeddedtissues. Immunofluorescence will be performed by using M30 CytoDEATHantibody (Boehringer Mannheim) with a fluorescence microscope (Axiophot,Zeiss). Scoring of apoptotic cells in these sections will be done byusing the OPTIMAS 6 software program (Optimas, Bothell, Wash.).Apoptotic index (%) will be calculated by dividing the number ofapoptotic cells (fluorescence positive) by the total number of cellscounted per cross-section of a sample of the prostate. Prostatepathological slides will also be processed by using antibodies for PCNA,and β-actin obtained from Santa Cruz Biotechnology forimmunohistochemistry assessment of tumor proliferation.

TUNEL Assay:

The ApopTag in situ apoptosis detection kit will be purchased fromIntergen (New York). In brief, paraffin-embedded tissue sections oftumors will be made from prostates harvested at sacrifice. Afterde-paraffinization of tissue section, apoptotic DNA fragments will belabeled by terminal deoxynucleotidyl transferase, and detected byanti-digoxigenin antibody that is conjugated to a fluorescein. Thesamples will be analyzed using a fluorescent microscope, equipped bydigital camera and the degree of apoptosis quantitated with AdobePhotoshop 5.5.

Statistical Design:

The study was designed in conjunction with the biostatistical core ofthe GCRC at UCLA and SPORE in prostate cancer. The following is anexample for most complex studies will be carried out with 4 groups in a2*2 design: (variable 1:) TRAMP vs LID TRAMP, (variable 2:) Saline vs.IGF-I antibodies infusion. Animals will be randomized: led into the fourtreatment groups by randomized permutated block design with variableblock length. There are two primary endpoints: prostate weight atsacrifice (including tumor) and pathology grade of tumor. At sacrificesecondary endpoints will include serum IGF-I, K-167, TUNEL. Sample sizecalculation assumptions: (1) The factorial analysis of variance hasthese effects: genotype and treatment and the interaction between thetwo. (2) No adjustment for the assessment of effects for a given primaryendpoint. (3) The responses of the tumors to the different treatmentswill be similar in magnitude. (4) The power analysis for each expectedor plausible effect size will be 0.85 and the sample size n is estimatedusing this power for each effect size. (5) From previous data there isevidence of a 30% effect on tumor weight during treatment. (6) Two-wayANOVA, a=0.05, 0.05, 0.15 LID and IGF-I AB effects, and theirinteraction, respectively. Powers for detecting the treatment effectslisted above.

This assumes that the tumor reduction for single treatment is 30%; twotreatments is 40%; then the power for detecting each effect is >0.95(a=0.05), for detecting the interaction of two treatments is 0.86(a=0.05) if N/group=10. The calculation therefore indicates thatN=10/group, 40 total will be the sufficient sample size for each study.

Statistical Analysis of Data:

The primary analysis for each of the two primary endpoints will beanalysis of variance for a 2*2-factorial design by estimation of each ofthe three effects in addition to F or T-tests. Also, correlated analysesbetween the primary endpoints will be studied in several ways includingANOVA. For longitudinal data, as secondary analysis, mixed-effectsmodels, using the SAS procedure PROC MIXED, and a generalized estimatingequations (GEE) approach, using the SAS procedure PROC GENMOD, will beexplored to study changes after treatments. In cases where modelassumptions such as normality or linearity are violated, non-parametricapproaches such as Wilcoxon-Mann-Whitney test, classification andregression tree will be explored to assess the association betweenoutcome variables and treatment exposure and other potential riskfactors for the outcomes.

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The contents of all references cited herein are incorporated byreference in their entirety.

What is claimed is:
 1. A method protecting cells in a subject from a DNAdamaging agent, the method comprising: starving the subject for 24-60hours to induce differential stress resistance in the subject; andadministering to the DNA damaging agent to the subject.
 2. The method ofclaim 1, wherein the subject is starved for 48 hours.
 3. The method ofclaim 1, wherein glucose level is reduced by 25 to 45 percent ascompared to the glucose level prior to starving of the subject.
 4. Themethod of claim 1, wherein IGF-I level in the subject is reduced ascompared to the IGF-I level prior to starving of the subject.
 5. Themethod of claim 1, wherein the DNA damaging agent is a DNA alkylatingagent.
 6. The method of claim 1, wherein the DNA damaging agent ismethyl methanesulfonate or menadione.
 7. The method of claim 1, whereinthe DNA damaging agent is methyl methanesulfonate, cyclophosphamide,etoposide or doxorubicin.
 8. The method of claim 1, wherein the DNAdamaging agent is etoposide or doxorubicin.
 9. The method of claim 1,wherein the subject is a cancer patient.
 10. The method of claim 1,wherein the subject is a prostate cancer patient.
 11. The method ofclaim 1, further comprising administering to the subject a cell growthinhibitor.
 12. The method of claim 11, wherein the cell growth inhibitorinhibits GH signaling in the subject.
 13. The method of claim 11,wherein the cell growth inhibitor is an IGFBP-1.
 14. A method ofprotecting a cell from a DNA damaging agent, the method comprising:starving the cell for 24-60 to induce differential stress resistance;and contacting the cell with the DNA damaging agent.
 15. The method ofclaim 14, further comprising contacting the cell with a cell growthinhibitor.
 16. The method of claim 15, wherein the cell growth inhibitoris an IGFBP-I.
 17. The method of claim 14, wherein the DNA damagingagent is a DNA alkylating agent.
 18. The method of claim 14, wherein theDNA damaging agent is methyl methanesulfonate or menadione.
 19. Themethod of claim 14, wherein the DNA damaging agent is methylmethanesulfonate, cyclophosphamide, etoposide or doxorubicin.
 20. Themethod of claim 14, wherein the DNA damaging agent is etoposide ordoxorubicin.
 21. A method protecting cells in a subject from a DNAdamaging agent, the method comprising: starving the subject with cancerfor 24-60 hours to induce differential stress resistance in the subject;administering to the DNA damaging agent to the subject; and monitoringthe toxicity of the DNA damaging agent in the subject.
 22. The method ofclaim 21, wherein toxicity of the DNA damaging agent is monitored bymeasuring LDH levels.
 23. The method of claim 21, wherein toxicity ofthe DNA damaging agent is monitored by measuring weight loss in thesubject.